"Dear Karbala, Dear Najaf, Dear Kadhimiyah and Dear Samarra, we warn the great powers and their lackeys and the terrorists, the great Iranian people will do everything to protect them," he said, naming the sites of the shrines in an emotive speech in Khoramabad, near the Iraq border.
Rouhani on Saturday pledged to help the Iraqi Prime Minister Nuri al-Maliki's government if it asked for assistance, though at that time no such request had been forthcoming.
In his speech on Tuesday, Rouhani mentioned petitions signed by Iranians who said they were willing to fight in Iraq "to destroy the terrorists and protect the holy sites", which are visited by hundreds of thousands of Iranian pilgrims annually.
"Thank God there are enough volunteers Shias, Sunnis and Kurds in Iraq to fight the terrorists," he added.
The Iranian pledges follow a call by top Shia cleric Grand Ayatollah Ali al-Sistani for Iraqis to volunteer to resist the onslaught spearheaded by Iraq and the Levant (ISIL), who hold the major cities of Mosul and Tikrit and are fighting north of the capital.
Maliki spent years in exile in Iran when dictator Saddam Hussein was in power in Baghdad.
The major Shia shrines in Iraq are in Najaf and Karbala, south of the capital, in the district of Kadhimiyah in Baghdad and in Samarra to the north, which the militants have made repeated, but so far unsuccessful, efforts to enter.
At least 5,000 Iranians have pledged online to defend Iraq's shrines against the extremists, a conservative news website in Iran reported on Tuesday.
40 Indians 'uncontactable'
Forty Indian employees stranded in violence-hit Iraq are "uncontactable", the foreign ministry said Wednesday, with a newspaper reporting the construction workers have been kidnapped. A ministry spokesperson said he could not confirm the report in the Times of India that insurgents have abducted the 40 workers in the northern city of Mosul amid a deteriorating security situation.
"Despite our best efforts at this stage we haven't been able to contact them. So they remain uncontactable at this stage," said spokesman Syed Akbaruddin told reporters.
"Beyond that I am not able to confirm or verify reports of a speculative nature (kidnapping)," Akbaruddin added.
"At this stage we have no reports whatsoever, no confirmation, no verification of any Indian national being involved in any violent accident or injury."
The Indian foreign ministry has set up a 24 hour control room in New Delhi to provide information on Iraq and was dispatching a former envoy to the country to assist its embassy in Baghdad.
The Times of India, citing unnamed sources, said the 40, who were working on various projects, were abducted by the militants during an evacuation of the Mosul area.
Since launching their offensive on June 9, ISIL has captured Mosul and a big chunk of Arab territory stretching south towards the capital.
As many as 46 Indian nurses were also stranded in Iraq waiting for the turmoil to subside. Several have told NDTV by phone that they were living like prisoners at a state-run hospital in Tikrit city after being abandoned by their employers as well as the military.
The offensive has displaced hundreds of thousands of people and sent jitters through world oil markets as the militants have advanced ever nearer to Baghdad, leaving the Shia-led government in disarray.
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Iran trapped badly now. if isis (saud-usa proxy) wins iraq next target is Iranian shias. if usa save iraq on request means free interventions in iran.
What have you done with the destruction of shrines in Saudi Arabia - probably nothing by the Saudi Government in the name of expansion. ISIL too is a Saudi-American proxy - it will be the people of Iraq standing next to their national army to safeguard the country and the shrines.
Something positive at last between the two waring countries Iran and Iraq.