Mr Nadezhdin has been relatively critical of Vladimir Putin’s full-scale war in Ukraine when few dissenting voices have been tolerated in Russia.
Election authorities claimed more than 15% of the signatures he submitted with his candidate application were flawed.
He had tried to challenge this, but the commission rejected his bid.
Refusing to give up, Mr Nadezhdin, 60, said on social media that he would challenge the decision in Russia’s Supreme Court.
The Central Election Commission said that of the 105,000 signatures submitted by Mr Nadezhdin, more than 9,000 were invalid and they cited a variety of violations.
That left 95,587 names, meaning he was just short of the 100,000 required signatures to register as a candidate, commission member Andrei Shutov said.
“There are tens of millions of people here who were going to vote for me, ” Mr Nadezhdin complained to the commission. “According to all polls, I am in second place after Putin.”
“The decision has been made,” declared commission chairwoman Ella Pamfilova. “If Nadezhdin wants, he can go to court,” Tass news agency quoted her as saying.
Russia’s presidential election is due to take place from 15-17 March, although the result is not in doubt as only candidates viewed as acceptable to the Kremlin are running.
A final decision on who can take part in the election will come on Saturday, but the election commission chairwoman said it was already clear there would be four candidates on the ballot.
Other than Vladimir Putin, they include nationalist leader Leonid Slutsky, parliament deputy speaker Vladislav Davankov and Communist Nikolai Kharitonov. All their parties have broadly backed Kremlin policies and none of the trio is seen as a genuine challenger.
“Running for president in 2024 is the most important political decision of my life. I am not retreating from my intentions,” Mr Nadezhdin wrote on Telegram. “I collected more than 200,000 signatures across Russia. We conducted the collection openly and honestly.”
Boris Nadezhdin is one of the few government critics whose voices have been heard on the ubiquitous talk shows on state-run TV since the invasion on 24 February 2022. He has appeared as a type of anti-war “whipping boy” that other guests would target for criticism.
In the 1990s he worked as an adviser for Putin critic Boris Nemtsov who was assassinated a stone’s thrown from the Kremlin in 2015. But he also has ties to Sergei Kiriyenko, a key Putin political overseer.
Although Mr Nadezhdin’s run for the presidency was viewed initially with suspicion by some opposition figures, Russia’s main opposition leader Alexei Navalny gave his backing to the Nadezhdin campaign from his jail cell inside the Arctic Circle, as did exiled former business magnate Mikhail Khodorkovsky.
Mr Nadezhdin appeared on the BBC last month promising to end the war in Ukraine on his first day as president, although he was realistic about his chances of success.
“My first task will be to stop the conflict with Ukraine, and then to restore normal relations between Russia and the Western community.”
He is not the first presidential hopeful to have run on an anti-war platform. In December, former TV journalist and independent politician Yekaterina Duntsova was barred from running because the election commission said there were mistakes on her application form.
Mr Nadezhdin said he had tapped into a wave of anti-war sentiment in Russia, meeting the wives of reservists who want their husbands to return from the war. His campaign started slowly and it was only in recent weeks that Russians began registering their support in large numbers.
His increasing success also attracted condemnation from pro-Kremlin propagandists such as Vladimir Solovyov, who suggested he might be a stooge for “Ukrainian Nazis”.