The foreign ministry handed "an official protest memorandum" to Mortaza Sanubari over "statements made by Ali Khamenei against the kingdom of Bahrain", BNA news agency said.
Khamenei said on Saturday that the nuclear deal reached between Tehran and world powers would not alter Iran's support for the governments of Syria and Iraq, nor its backing for "oppressed people" in Yemen and Bahrain, and the Palestinians.
Iran has provided money and military advisers to support President Bashar al-Assad's regime in Syria against rebels it accuses Gulf Arab states of arming with Western connivance.
Riyadh, the region's main Sunni power, has in turn accused its Shiite rival Iran of malign military intent and meddling -- charges shared by other Gulf Arab states.
Bahrain's foreign affairs undersecretary, Abdulla Abdullatif Abdulla, said Khamenei's statements "represented a flagrant and unacceptable interference in Bahrain's internal affairs", BNA reported.
He called on Iran to put "an end immediately to such statements " and respect "the principles of good neighbourliness and non-interference in the internal affairs" of Bahrain.
Home to the US Fifth Fleet and strategically situated across the Gulf from Iran, Bahrain has witnessed sporadic demonstrations since it crushed a protest movement in 2011.
At least 89 people have been killed in clashes with security forces since 2011, while hundreds have been arrested and put on trial, rights groups say.
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