"No compromise, no surrender" and "Get out of the NPT", the mostly-male crowd of about 1,500 chanted during a brief march after weekly Muslim prayers at Tehran University.
The protest followed a sermon by former president Akbar Hashemi Rafsanjani in which the influential figure and reputed pragmatist did not refer to Iran's pledge to bow to International Atomic Energy Agency (IAEA) demands.
On Tuesday, during an unprecedented mission here by the foreign ministers of Britain, France and Germany, the Islamic republic agreed to sign an additional protocol to the NPT that allows tougher checks by the UN's nuclear watchdog.
And just 10 days before the expiry of an IAEA deadline for Iran to come clean on its suspect nuclear programme, the country also agreed to make a full declaration of its activities as well as to suspend uranium enrichment.
Iran's failure to meet the IAEA deadline could have seen it declared in breach of the NPT, and the matter referred to the UN Security Council.
The deal has been seen as a capitulation by many Islamic hardliners, who have maintained calls for their leadership to follow the path of North Korea and pull out of the NPT altogether.
The hardline camp sees the signing of the additional protocol as tantamount to a violation of the national sovereignty of Iran, which insists its nuclear programme is aimed solely at generating atomic energy.