Six US-allied Gulf Arab states demanded on Tuesday that Iran end what they called interference in the region, reiterating a long-held mistrust of their main rival.
The Islamic Republic denies trying to subvert Saudi Arabia and its wealthy Gulf neighbors.
A communique issued at the end of a two-day summit of the Saudi-led Gulf Cooperation Council (GCC) also urged action to halt mass killings and violations of international law in Syria.
The oil-producing GCC states wield influence out of proportion to their sparse populations due in part to global energy and investment links, generous international aid and Saudi Arabia's role as home to Islam's two holiest sites.
"The council expressed its rejection and condemnation of the continuing Iranian interference in the affairs of the Gulf Cooperation Council's states and called on Iran to stop these policies," the communique said.
On the conflict in Syria, the statement, read out by GCC Secretary General Abdulatif al-Zayani, added: "We ask the international community for serious and swift moves to stop these massacres and these severe attacks that contradict all religions and international laws and humanitarian principles."
The GCC called on the international community "to provide all forms of urgent humanitarian aid" for the people of Syria.
Kuwait said on Monday it would host an international humanitarian donor conference for Syria at the end of January, amid growing concern for millions of Syrians suffering war, homelessness and winter cold.
Gulf Arab leaders have long called for Syrian President Bashar al-Assad to step down, and in November the GCC recognized a newly-formed opposition coalition as the legitimate representative of the Syrian people.
The communique did not elaborate on Iran, but the most common Gulf Arab complaint about alleged Iranian meddling in the region relates to Bahrain, which has repeatedly accused Tehran of interference in its internal politics.
Iran sees the Gulf as its own backyard and believes it has a legitimate interest in expanding its influence there.
Bahraini Foreign Minister Khalid Bin Ahmed Bin Mohamed al-Khalifa told reporters Iran posed a "very serious threat."
"Politically, (there is) lots of meddling in the affairs of GCC states; an environmental threat to our region from the technology used inside nuclear facilities; and there is of course the looming nuclear program," he said, referring to Iran's disputed atomic work.
"So the threat level is quite high, but we are ready if faced with circumstances that require action."
While not racked by disturbances on the scale of Egypt or Libya, Bahrain has been volatile since pro-democracy protests led by its Shia Muslim majority erupted last year.
Bahrain's Sunni Muslim rulers brought in Saudi and United Arab Emirates forces last year to help quell the protests, and Shia power Iran condemned the move, saying it could lead to regional instability. Bahrain has accused Iran of being behind the unrest. Tehran denies this.
Bahrain's Shias complain they have long been marginalized in political and economic life, an assertion the government denies. Its Sunni rulers have rejected the protesters' main demand for an elected government.