"That (the) US arms a terrorist group is what causes instability," Khameini wrote on Twitter Monday. "Who created ISIS? The US!"
He added that while US President Donald Trump accuses Iran of supporting terrorists, "terrorism in this region has American roots."
Trump last week responded to terrorist attacks in the Iranian capital of Tehran by warning "states that sponsor terrorism risk falling victim to the evil they promote," a reaction described as "repugnant" by Iran's foreign minister.
Those attacks, which left at least 12 people dead, were claimed by ISIS.
Iran's Islamic Revolutionary Guards, a powerful branch of the country's military, vowed revenge for the attacks and blamed them on longstanding regional rival Saudi Arabia.
ISIS has its roots in al Qaeda in Iraq, which took part in the insurgency that followed the 2003 US-led invasion of the country.
While Saudi Arabia has been threatened by the group, officials in the region and their Western counterparts have claimed ISIS receives funding from backers inside the Kingdom as well as from other rich Middle Eastern countries.
Khamenei's comments come as tensions in the Middle East are among their highest in years, following the cutting of ties between major regional powers led by Saudi Arabia, and Qatar, which has been accused of supporting terrorism in the region.
Qatar has been ejected from the Saudi-led coalition fighting Houthis in Yemen, and transport links between it and three other Gulf countries have been severed, with Qataris living in Saudi Arabia, Bahrain and the UAE ordered to leave. Turkey and Iran have stepped into the gap, raising the specter of a reshuffling of alliances in the Gulf region.
Qatar's Foreign Minister Mohammed bin Abdulrahman Al Thani said Monday that the country's government still has "no clue what are the main reasons behind all these measures."
Saudi Arabia, the UAE and Bahrain accuse Qatar of supporting terrorism networks in the region. UAE Foreign Minister Anwar Gargash, and key architect of the blockade on Qatar told CNN's John Defterios that Qatar offers "very, very huge logistical, financial support for extremist groups."
"There is no more trust," he said, adding, "it is time for cooler heads to restructure Qatar's approach on foreign policy."
Gulf leaders have also been critical of Qatar's relatively neutral stance on Iran, which they view as a prime destabilizing force in the region.
The diplomatic rift came two weeks after Saudi Arabia, the UAE, Bahrain and Egypt blocked several Qatari media outlets, including Al Jazeera, over comments allegedly made by Qatari Emir Sheikh Tamim bin Hamad Al Thani.
Al Thani reportedly hailed Iran as an "Islamic power" and criticized US President Donald Trump's policy towards Tehran. Qatar said the official news agency which reported the comments was hacked -- and on June 6, US officials told CNN that US investigators believe Russian hackers were behind it.
Two large regional powers on either side of the Sunni-Shiite Islamic sectarian divide, Saudi Arabia and Iran have been engaged in a longstanding cold war, tussling for influence in countries in their sphere of influence, especially Syria, Lebanon and Yemen, where Saudi-led bombing has left thousands of civilians dead.
Qatar and Iran share the largest underwater natural gas field in the world. But recent Gulf reports have charged the relationship goes beyond resource management, accusing Qatari officials of meeting with the head of Iran's Revolutionary Guard Corps.
But al Thani disputed that Monday, saying that "if the problem is Iran, why have those measures been taken against Qatar, why not taken against Iran?"
Seeking a solution
Both Qatar and Saudi Arabia are major US allies, and have close ties to European powers. But while Trump has signaled support for Riyadh despite efforts by the Pentagon and State Department to remain neutral, his European counterparts have been active in trying to tamp down tensions.
Al Thani praised French President Emmanuel Macron Monday for being "very active" in attempting to find a "solution to the problem."
British Foreign Secretary Boris Johnson, who is currently in the Gulf for a series of meetings, on Monday "urged all sides (to) refrain from any further escalation and to engage in mediation efforts."
Johnson encouraged Qatar to "take seriously their neighbors' concerns" but also expressed alarm at the blockading of the country and called for it to be eased.
"Qatar is a partner of the UK in the fight against terrorism but they urgently need to do more to address support for extremist groups, building on the steps they have already taken," Johnson said.