"The United States must know its interests and security in the region are in danger and that it cannot escape the consequences of this great crime," he said in an hour-long telephone call with Macron, quoted in a statement by the Iranian presidency.
Friday's killing of General Qasem Soleimani in a US drone strike in Baghdad on the orders of President Donald Trump ratcheted up tensions between the arch-foes.
Khamenei vows revenge for Soleimani killing through direct attack on US interests
Iran vowed "severe revenge" before Trump warned that Washington had lined up 52 targets if Tehran attacked American personnel or assets.
Rouhani said the Americans had "committed a major strategic error in assassinating General Soleimani", head of the Revolutionary Guards' foreign operations arm.
The Iranian president added that the killing might have the opposite effect of what the US had expected.
"This crime has strengthened as never before the unity and solidarity of the Iranian people, and also that of the Iraqi people," he told Macron.
"The Islamic Republic of Iran never seeks war and insecurity in the region, but it does not hesitate to defend its rights and sovereignty," he said.
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