The comments come ahead of meeting later this month between his Foreign Minister Mohammad Javad Zarif and EU foreign policy chief Catherine Ashton on restarting negotiations on the Islamic republic's controversial nuclear ambitions.
"Our government will not give up one iota of its absolute rights" on the nuclear issue, said Rowhani, a reputed moderate, repeating a mantra frequently used by his predecessor Mahmoud Ahmadinejad.
Soon after his election as president in June, Rowhani said he wanted "serious" talks with world powers to resolve Western suspicions that Iran's nuclear drive is cover to build a bomb despite repeated denials by Tehran.
That has led to several rounds of international sanctions being slapped on the Islamic republic, crucially targeting its financial and oil sectors, choking the economy and stoking a raging inflation.
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The last round of talks between Iran and the so-called P5+1 of world powers ended in Almaty in April with an impasse.
In parallel diplomatic efforts, the International Atomic Energy Agency (IAEA) is urging Iran to provide necessary cooperation to remove suspicions it seeks a nuclear weapons capability.
"Given the nature and extent of credible information available to the agency about possible military dimensions to Iran's nuclear programme, it remains essential and urgent for Iran to engage with us on the substance of these concerns," IAEA chief Yukiya Amano said yesterday.
Himself a former nuclear negotiator, Rowhani today did not elaborate on efforts to resolve such concerns over Iran's nuclear programme, on which all final decisions -- like other key issues in the Islamic republic -- rest with the country's supreme leader, Ayatollah Ali Khamenei.
Rowhani added that after the Zarif-Ashton meeting in New York, nuclear negotiations would "continue in another place with the 5+1 group," which groups the United States, Russia, China, France, Britain and Germany.