The rally came two days after parliament passed a law banning the denial of atrocities committed by the hardline communist regime -- a move which the opposition claimed was politically motivated before elections in July.
In a recording posted on a government website last month, the deputy head of the Cambodia National Rescue Party (CNRP), Kem Sokha, purportedly said that Tuol Sleng prison in the capital Phnom Penh was staged by Vietnamese soldiers who ousted the Khmer Rouge in 1979.
Survivors from Tuol Sleng, also known as S-21, urged Kem Sokha to apologise as protesters gathered in a park in Phnom Penh before marching to the headquarters of the CNRP.
"I won't allow anyone to distort history while I'm alive. We demand that Kem Sokha lights incense sticks and apologises before the souls of the dead," said 83-year-old survivor Chum Mey, who led the protest.
Local media reported thousands of people also came out in many provinces across the country today to protest at the remarks.
The former head of the prison, Kaing Guek Eav, better known as Duch, was sentenced last year to life in jail.
Protesters carried banners reading: "Kem Sokha is the first person who dares to insult the souls of all victims from Pol Pot's regime" and "Kem Sokha is more cowardly than Duch".
"I feel very hurt and I am angry with what he said," said Nov Sorn, 61, who lost her husband, father and a brother under the communist regime, which oversaw the deaths of an estimated two million people.
His main opponent Sam Rainsy is barred from running due to convictions which he contends are politically motivated.
Under the new law, which was approved Friday by a parliament controlled by lawmakers from Hun Sen's ruling party, anyone denying Khmer Rouge atrocities risks a prison sentence of up to two years.
Led by "Brother Number One" Pol Pot, who died in 1998, the Khmer Rouge from 1975-79 wiped out nearly a quarter of Cambodia's population through starvation, overwork or execution in a bid to create an agrarian utopia.
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