Officials in the city of Novosibirsk ruled that Parnas, the main opposition coalition, had failed to collect enough valid signatures to take part.
Russian opposition leader Alexei Navalny called the move a "blow to the democratic freedom of Russians".
The regional ballot is seen by Russia's opposition as a key test in the run-up to next year's parliamentary vote.
The three hunger strikers were detained by police and fined for staging a protest.
They and other activists in the Siberian city had spent weeks collecting the thousands of signatures required to register for election.
But the election commission in Novosibirsk - Russia's third largest city - found faults with more than 1,000 of the 11,000 signatures they submitted.
The activists insist they were valid, and said the authorities used out-of-date databases to check them.
In one case, a woman's signature was ruled out because her middle name came up in records as Timurovich, a man's name, instead of Timurovna.
The three - Leonid Volkov, Sergey Boyko and Yegor Savin - have said they will eat nothing and drink only water until their case is reviewed.
Mr Navalny warned that the "surreal" action could be replicated in other elections, both regional and national. He is a fierce critic of President Vladimir Putin, accusing the government of large-scale corruption.
"There is a political order not to let the opposition take part in elections," he told Moscow Echo radio station.
Three Russian opposition activists have gone on hunger strike after they were barred from running in local elections.