When the head of the jury announced that it had found the three men "not guilty" of involvement in the journalist's murder, the Makhmudov brothers hugged each other while their supporters shouted "bravo" and "thank you".
A former FSB (Federal Security Service) agent, Pavel Ryaguzov, was also acquitted by the jury of an extortion charge relating to the case.
Speaking outside the court, one of the men's lawyers, Murad Musayev, called the decision a "victory" for Russian justice.
"The only way to stop these crimes is to find the real criminals," he said.
A lawyer for Ms Politkovskaya's family, Karina Moskalenko, called on the security services to "start a proper investigation".
The defendants were allowed to leave the court free men
"We demand, we need the real killer, the real killers, and we shall achieve this," she said.
State prosecutor Yulia Safina said the prosecution would appeal against the verdict.
The suspected gunman, Rustam Makhmudov, 34, is believed by investigators to be hiding in Western Europe.
They have not named or arrested anyone suspected of ordering the killing.
The Makhmudov brothers come from Chechnya, where Ms Politkovskaya did some of her most damning reports into alleged abuses by Russian security forces.
The BBC's Rupert Wingfield-Hayes in Moscow says that for the three men accused of taking part in Ms Politkovskaya's murder, this result is a huge relief.
This failure amounts to a human rights crisis
Miklos Haraszti, OSCE
But for the Russian authorities, it is a public relations disaster, our correspondent says.
This trial was supposed to show justice would be done - instead it has turned into a fiasco, he adds.
Our correspondent says the evidence against the men was extremely weak and in the end the jury simply did not believe the prosecution.
When asked about the acquittals, the chairman of the Russian Union of Journalists, Vsevolod Bogdanov, said he was "ashamed".
"I have this feeling of incredible shame - at what level was the investigation conducted that the jurors delivered this verdict unanimously?" he told the Interfax news agency.
The media freedom representative for the Organisation for Security and Co-operation in Europe (OSCE), Miklos Haraszti, told the Reuters news agency that Russia's failure to solve the murder amounted to a "human rights crisis".
Last month, a journalist for Novaya Gazeta, Anastasia Baburova, was shot dead along with prominent human rights lawyer Stanislav Markelov on a street in central Moscow.
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