Bahrain's foreign ministry has summoned Iran's charge d'affaires in Manama to protest at what it described a "gross violation of its sovereignty".
It follows official Iranian calls for demonstrations against a proposed union between Saudi Arabia and Bahrain.
A summit of the Gulf states discussed the proposal on Monday but did not reach any decision on the matter.
Bahrain's Sunni royal family has accused Iran of supporting mass protests by the nation's Shia majority.
The proposed union was presented as a first step in a larger integration between all six Gulf Co-operation Council (GCC) members.
It calls for economic, political and military co-ordination and a new decision-making body based in Riyadh, replacing the current GCC Secretariat.
After no agreement was reached, the Saudi foreign minister said the discussions were being postponed.
But the mere suggestion of the union sparked strong criticism from the opposition in Bahrain.
The tiny Gulf island has seen intermittent protests for more than a year demanding political reforms.
The conflict reached a turning point when Saudi Arabia led a military force into Bahrain as part of what was known the "Peninsula Shield Forces" to support the authorities.
The withdrawal of the troops became a constant demand by the opposition.
Earlier this week, Iranian Parliament Speaker Ali Larijani was quoted by the official Irna news agency as saying: "If Bahrain is supposed to be integrated into another country, it must be Iran and not Saudi Arabia."
The Islamic Propagation Co-ordination Council, which organises state-backed protests, has called for rallies after Friday prayers against what it described as a US plan to annex Bahrain to Saudi Arabia.
Iranian Foreign Ministry spokesman Ramin Mehmanparast warned that "any kind of foreign intervention or non-normative plans without respecting people's vote will only deepen the already existing wounds".
Bahrain's foreign ministry condemned the comments, saying they "represent a flagrant interference in the internal affairs of the kingdom, and gross violation of its sovereignty and independence, and they constitute a completely unacceptable conduct".
The GCC was formed in 1981 as the Sunni-dominated monarchies of the Gulf aimed to bolster security after the 1979 Islamic revolution in Iran and to counter the ambitions of then Iraqi President Saddam Hussein.