Khamenei endorsed Ahmadinejad's victory in the June 12 election, which was followed by opposition protests and plunged Iran into its deepest internal crisis since the 1979 Islamic revolution.
Khamenei said the election was held and there was a big voter turnout but that some people had "unfairly" ignored this.
"The leader … termed questioning the basis of the election the biggest crime," state television said in a report from Khamenei's meeting with leading scientists in Tehran.
It quoted him as saying: "The day after the election, some people called the glorious election a lie. Is this a minor crime?"
The authorities have portrayed the post-election street demonstrations, which were quelled by the elite Revolutionary Guards and an allied Islamic militia, as a foreign-backed bid to undermine the Islamic Republic.
"Of course some people inside (Iran) may not be aware that they are moving in line with the enemies' threats, but this issue will not change the truth," said Khamenei, who has final say on all matters of state.
Thousands of people were arrested after the presidential election, which the authorities have described as the "healthiest" vote Iran has held in the last three decades.
Most of the detainees have since been released, but more than 100 senior reformers, activists, journalists and others, have been put on trial accused of fomenting unrest.
(Reporting by Reza Derakhshi and Hossein Jaseb; writing by Fredrik Dahl; editing by Janet Lawrence)
