Chanting "Peace be upon (Prophet) Muhammad and his family", the opposition supporters, many dressed in black, marched in south Tehran, the witnesses said.
Mousavi had urged his supporters to wear black as a sign of remembrance and remain peaceful.
Al Jazeera's Alireza Ronaghi, reporting from Tehran, said protesters from various walks of life had joined the march on Thursday "to get their votes back".
"People were sad, they were quiet, but they were determined. That's what we hear from the people who have been in the rally," he said.
"I have also talked to others, they say that the quiet mood ruling over all the marches has gone to a new height recently."
One witness said the marchers carried pictures of Mousavi and placards like "We have not had people killed to compromise and accept a doctored ballot box" and "Silent, keep calm".
Mousavi somehow managed to reach the venue and addressed the huge crowd.
He announced that a rally scheduled for Friday had been cancelled, and that his supporters should prepare for a major march planned for Saturday afternoon from Tehran's Revolution Square to Freedom Square.
Mousavi has applied for a permission at the interior ministry but it is unclear whether this would be issued.
Discussing Mousavi's options, Sadegh Zibalalam, who teaches politics at the University of Tehran, told Al Jazeera: "So far he has been very determined and defiant.
"In every speech that he has made during the past five days, he has assured his followers that he is not going to back down, that he is going to stand firm, and he doesn't the accept the election results.
"He has been very, very determined and very dependable."
About 100 people gathered outside the United Nations building in Tehran earlier on Thursday urging the Guardian Council to take action over the disputed poll.
Officials have barred the foreign media from covering such "unauthorised" events.
However, they are expected to ensure a heavy turnout for a special sermon to be delivered by Ayatollah Ali Khamenei, the country's supreme leader, at the Tehran university campus on Friday.
The organisers have invited all Iranians to attend to show solidarity and unity.
Responding to the ongoing protests against the declaration of Ahmadinejad as the election winner, the authorities have announced a series of concessions.
In the latest move, a spokesman for the Guardian Council said on Thursday that the 12-member body would meet the candidates on Saturday.
The body had already begun the "careful examination" of a total of 646 complaints submitted in connection with the vote, Abbas-Ali Kadkhodaie said.
|Restrictions on coverage of protests has forced the world media to rely on amateur images
"This will enable them to raise issues and points they wish to discuss with the members of the council, and also provide a direct contact with the candidates," he said.
Besides Mousavi, the two other candidates contesting the outcome are Mehdi Karoubi, a reformist, and Mohsen Rezaie, a conservative and former head of Iran's Revolutionary Guards.
Ahmadinejad was also expected to be invited to the meeting.
The Guardian Council has already said it may order a partial recount of the ballots, but will not annul the result.
Zibakalam told Al Jazeera: "In the past, [Khamenei] has come under some criticism for attaching himself too much to Ahmadinejad and supporting him uncritically and unreservedly.
"Against that background, it will be very difficult for [Khamenei] to make a U-turn and to declare the election null and void."
A Mousavi supporter in Tehran told Al Jazeera that he believes a recount, if done properly, could be good for the country.
"Pursuing things through legal means is the best for everyone. What should not be undermined here is the security of the country and the society," he said in a phone interview.
"I do believe [a recount] would be very healthy if done under certain measures and if done by the consent of the nominees and the candidates of the defeated parties.
"What I really desire for my country is peace and security above all. I really look forward to a situation where all parties can come into a very peaceful and understanding conclusion."
Tension between the opposition and supporters of the government could rise in the coming days if the Guardian Council's moves fail to resolve the election dispute.
Authorities have shut down internet sites and mobile telephone text services in an attempt to bring the protests under control.
Despite these measures, violent scenes of police beating Mousavi supporters taken on mobile phones have been broadcast on news bulletins across the world, though the authenticity of such footage often cannot be verified.
"University cities in Iran have always been very active in political dissent," Anoushaka Maraslian, a Middle East analyst in London, told Al Jazeera.
"That's the concern of the elders; that's the concern of the Guardian Council, and that's why they are making conessions, because they realise that young Iranians are leading the protests ... with parallels to [the revolution in] 1979."