Khamenei is officially the imam of Tehran but usually delegates the task of leading Friday prayers to others.
The last time he led Friday prayers at Tehran's Mosalla mosque was in February 2012, on the 33rd anniversary of the Islamic revolution and at a time of crisis over the Iran nuclear issue.
Meanwhile, President Hassan Rouhani appealed Wednesday for "unity" and flagged the need for radical changes to the way Iran is run, after a wave of angry protests over the accidental downing of a Ukrainian airliner.
The Kiev-bound Boeing 737 was shot down in a catastrophic error shortly after takeoff from Tehran, killing all 176 people aboard.
One week on from the disaster, Rouhani called for "national unity" in remarks broadcast live on state television.
"If there was a delay" by the armed forces to release information about what happened, "let them apologise," he said.
Rouhani also sought a full explanation after it took Iran days to admit the airliner had been mistakenly shot down.
The blunder sparked four consecutive nights of protests in Tehran calling for those responsible to resign or be prosecuted, leading to at least 30 arrests.
Iranian Foreign Minister Mohammad Javad Zarif said they were demonstrating after being deceived for days.
"In Iran... people can state their views. Sometimes they state their views in excessive ways, sometimes the reaction is excessive," he said in New Delhi.
"But the fact of the matter is, (over the) last few nights we've had people in the streets of Tehran, demonstrating against the fact that they were lied to for a couple of days."
Iran has been rocked by crisis after crisis since 2018.
In May that year, US President Donald Trump unilaterally withdrew from a landmark 2015 agreement that gave Iran relief from sanctions in return for curbs on its nuclear programme.
Since then, the US has relentlessly slapped waves of sanctions on Iran as part of a "maximum pressure" campaign.
Washington says the campaign is aimed at reining in Tehran's missile programme as well as its "destabilising behaviour" in the region.
Iran has hit back by progressively reducing its nuclear commitments.
On Tuesday, as tensions mounted over the downed airliner, European parties to the deal launched an arbitration process charging Iran with failing to observe its commitments to the accord.