The conservative cleric, Iran's highest authority, has refrained from making decisive statements on the nuclear deal reached last month, which must still be approved by the US Congress before taking effect.
Read: Kerry seeks to convince Gulf Arabs on Iran deal
"They thought this deal - and it is not clear if it will be passed in Iran or in America - will open up Iran to their influence," Khamenei was quoted on his website as saying at a meeting with members of the Islamic Radio and Television Union.
"We blocked this path and will definitely block it in the future. We won't allow American political, economic or cultural influence in Iran."
Read: Blows for Obama as key lawmakers come out against Iran deal
Most analysts see the chance of Khamenei rejecting the deal as small after he gave President Hassan Rouhani the political cover to pursue talks. But he also holds a deep mistrust of the United States, which he still refers to as the 'Great Satan'.
If the deal is implemented, international sanctions on Iran will be lifted, opening up a market of nearly 80 million people to foreign investment after years of isolation.
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