In a fast-moving sequence of events following Crimea's controversial secession referendum on Sunday, the Kremlin said Crimea was now considered part of Russia and no longer Ukrainian territory, shrugging off strong international objections.
"In the hearts and minds of people, Crimea has always been and remains an inseparable part of Russia," Putin said in an emotional address broadcast on television.
The move, which came sooner than expected, risks plunging the West and Russia into a crisis not seen since the Cold War and sent shock waves through the new authorities in Ukraine who took over after the ousting of pro-Kremlin president Viktor Yanukovych last month.
Putin signed the treaty with Crimean Prime Minister Sergei Aksyonov and other Crimean leaders at a ceremony at the Kremlin attended by both houses of parliament.
Lawmakers, who still have to formally ratify the treaty, broke into applause and cheers after the signing.
"The Republic of Crimea is considered to be part of Russia from the date of the signing of the treaty," the Kremlin said.
Crimea and the city of Sevastopol - the home of the Russian Black Sea fleet which has special status - are being incorporated as new constituent parts of the Russian Federation.
The signing - which had not been flagged in advance - came after Putin gave a fiery address at the Kremlin seeking to justify the incorporation of Crimea into Russia.
His defiant speech brushed off US and EU sanctions touted as the most severe against Moscow since the end of the Cold War.
Putin said Crimea belonged with Russia and he slammed the Soviet-era decision by Nikita Khrushchev to gift the peninsula to the Ukrainian Soviet republic as riddled with "violations".
"When Crimea suddenly ended up being in another state, Russia felt it was not simply robbed… it was plundered," he said.
He added that Russia was tired of being pushed into a corner by the West and said it had been repeatedly deceived on issues like NATO, missile defence and visa-free travel.
"On Ukraine the West crossed a line," he said, warning it against provoking Russia. "They are trying to drive us into a corner."
But he sought to play down fears that Russia was seeking to also incorporate parts of eastern and southern Ukraine.
"We don't want the break-up of Ukraine. We do not need it," Putin said.
The seizure of Crimea by pro-Russian forces following the ouster of Yanukovych last month has been condemned around the world.
But the prospect of Russia's international isolation did not deter an increasingly defiant Putin who based the justification for the de-facto annexation of Crimea on the region's weekend referendum where almost 97 percent voted to split from Ukraine.
It is the first time since World War II that Moscow is expanding its borders and represents the most radical redrawing of the map of Europe since Kosovo's 2008 declaration of independence from Serbia.
The lush peninsula has been the base of Kremlin navies since the late 18th century and only became part of Ukraine in 1954 when it was gifted by Khrushschev.
With a population of some two million, Crimea has long been seen as a favourite vacation destination and has a great hold over the Russian psyche.
After the treaty was signed, the party of Ukrainian boxer-turned-politician Vitali Klitschko urged Kiev to cut diplomatic ties with Russia.
Patriotic fervour has soared in Russia, with tens of thousands expected to hold a state-supported rally in Moscow with the slogan "We are together".
Independent pollster Levada Center said Monday that two thirds of Russians -- 63 per cent -- believe Russia is a "great nation," double the figure of 31 percent in March 1999.
Putin's approval ratings have jumped to 72 per cent on the back of the success of the Sochi Winter Olympics and his decision to send troops to Crimea following Yanukovych's February 22 ouster.
Russian Deputy Prime Minister Dmitry Rogozin has ridiculed sanctions against him on Twitter and parliament lawmakers adopted a statement urging Washington and Brussels to blacklist them too.
A defence ministry source told AFP that there was relief within the navy that the future was now assured of the Russian Black Sea fleet at a Crimea base that Moscow was leasing from Ukraine.
But the mood was far from jubilant among economists and businesses who fear Putin's policies and the threat of further sanctions will inflict lasting damage on Russia's economy.
US President Barack Obama on Monday slapped sanctions on seven Russian officials and four Ukrainians accused of usurping Ukraine's territorial integrity, including Yanukovych.
"If Russia continues to interfere in Ukraine, we stand ready to impose further sanctions," Obama warned.
The European Union unveiled travel bans and asset freezes against 21 Russian and Ukrainian officials, including Vice Admiral Alexander Vitko, the head of Russia's Black Sea fleet.
"These are by far the most comprehensive sanctions applied to Russia since the end of the Cold War -- far and away so," said a senior US official.
Those targeted under Obama's executive order will see assets and interests in the United States or under US jurisdiction blocked and Americans will be barred from doing business with them.
Diplomats in Brussels said EU and Ukrainian leaders would on Friday sign the political portion of a landmark pact whose rejection by Yanukovych in November sparked the protests that led to his fall.
Ukraine says will never recognise Crimea integration into Russia
Ukraine will never recognise Russia's annexation of Crimea, a spokesman for the foreign ministry in Kiev said Tuesday, quoted by news agency Interfax-Ukraine.
"We do not recognise and will never recognise the so-called independence and so-called agreement on Crimea's integration into Russia," said Yevgen Perebyinis, adding that the signed accord had "nothing to do with democracy, rule of law or common sense."
Russia's Crimea grab 'against international law': Merkel
German Chancellor Angela Merkel said Tuesday that Russia's absorption of Ukraine's Crimea region after a secession referendum and declaration of independence there violated international law.
"The so-called referendum breached international law, the declaration of independence which the Russian president accepted yesterday was against international law, and the absorption into the Russian Federation is, in our firm opinion, also against international law," she said.
UK suspends military cooperation with Russia: Hague
Britain will suspend all bilateral military cooperation with Russia after it signed a treaty making Crimea a Russian territory, Foreign Secretary William Hague said on Tuesday.
"We have suspended all such cooperation," Hague told parliament, saying a technical cooperation agreement and joint naval exercises with Russia, France, Britain and the United States had been put on hold.
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The bear has broken up again.
Russia is too strong to fear isolation. Plus it's never isolated. India,china,iran have good relations with it and will continue to have so irrespective of what Western country thinks.