Iran's Khamenei backs Rouhani's New York talks
"We support the diplomatic initiative of the government and attach importance to its activities in this trip," he said on his website.
But he added that some of what had occurred "was not appropriate".
Mr Rouhani had spoken to US President Barack Obama by phone in a bid to ease tension over Iran's nuclear programme.
They were the first talks between Iranian and US leaders in more than three decades.
Iran wants the lifting of UN sanctions, which were imposed over concerns that it is seeking to build nuclear weapons clandestinely - an allegation Tehran denies.
Israeli Prime Minister Benjamin Netanyahu, the leading advocate of a hard line against Iran over its nuclear programme, has dismissed Mr Rouhani as a "wolf in sheep's clothing".
'No trust'
When Mr Rouhani returned from New York he was greeted at Tehran airport by supporters but also met hardline protesters chanting "Death to America".
In his comments on Saturday, Ayatollah Khamenei said that "some of what happened on the New York trip was not appropriate", without being specific.
"We are pessimistic towards the Americans and do not put any trust in them," he said.
"The American government is untrustworthy, supercilious and unreasonable, and breaks its promises."
Substantive nuclear negotiations between Iran and international powers are due to take place on 15 October and Mr Rouhani has promised to bring a plan to the meeting.
He said he wanted to reach a deal over the nuclear issue in three to six months.
In an interview conducted on Friday, President Obama told the Associated Press that the US believed Iran was still "a year or more away" from building a nuclear weapon.
He also expressed optimism about the current diplomacy.
"Rouhani has staked his position on the idea that he can improve relations with the rest of the world," Mr Obama said. "And so far he's been saying a lot of the right things. And the question now is, can he follow through?"
BBC
