The protesters chanted "Russia without Putin" and "New elections, New elections" as speakers called for an end to Putin's 12-year domination of the country at the second big opposition rally in two weeks in central Moscow.
"Do you want Putin to return to the presidency?" novelist Boris Akunin asked from a large stage.
Whistling and jeering, protesters chanted: No!"
Police said at least 28,000 attended the rally on Prospekt Sakharova (Sakharov Avenue), named after Soviet-era dissident Andrei Sakharov. One opposition leader put the crowd size at up to 100,000 people.
The big turnout is likely to encourage organizers to believe they can keep up the momentum of the biggest opposition demonstrations since Putin rose to power 12 years ago, although the prime minister seems intent on riding out the protests.
Tens of thousands of people protested in Moscow on December 10 and many more demonstrated across the world's largest country the same day to complain against alleged vote-rigging in the December 4 election won by Putin's United Russia party.
The protesters were heartened by the Kremlin's human rights council saying a new election should be held, although it is only an advisory body whose recommendations are regularly ignored by Russia's leaders.
Many of the protesters wore white ribbons, the symbol of the protests, and others carried balloons and flags at the rally, which brought together liberals, nationalists, anarchists, environmentalists and urban youth on a bitterly cold day.
"The last protest made a huge impression and I want others to come and realize they can stand up for their right. We all know the election result and we all know how dishonest they were," said Andrei Chernyshov, a 22-year-old student.
PROTESTERS DISMISS CONCESSIONS
President Dmitry Medvedev, who is stepping aside next year to let Putin take his place after four years as prime minister, has promised electoral reforms to relax the Kremlin's grip on power, including restoring the election of regional governors.
But the opposition has rejected these conciliatory efforts and says Putin and Medvedev have ignored its key demand for a rerun of the parliamentary poll, which handed a slim majority to the ruling United Russia party.
The protesters say United Russia benefited from widespread voting irregularities and international monitors said the vote was slanted in the ruling party's favor.
"The party of swindlers and thieves are the only ones who benefit from the preservation of the status quo," journalist Leonid Parfyonov said on a video message shown on a screen.
Protesters held signs saying: "For Russia without Putin."
Dozens of police trucks lined the capital's main ring road nearby and the police blocked off roads around the protest site, but they did not intervene.
Putin is still expected to win the presidential election in March and return to the post he held from 2000 until 2008 but there are growing doubts that he will win outright in the first round of voting. The opposition is largely divided and has no candidate to unite it in the March presidential poll.
The former KGB spy's popularity has declined since he and Medvedev announced plans in September to swap jobs next year, a decision which many Russians said showed a disregard for democracy.
Putin, 59, has suggested that many of those taking part in the protests have been paid to turn out and accused the United States of encouraging the protests. Many people, who answered calls to protests on social networking sites, say this underlined that he is out of touch.