Speaker Yael Braun-Pivet, who is from President Emmanuel Macron's centrist party but is officially neutral, confirmed she would reject on constitutional grounds the bid to introduce new legislation, infuriating its backers.
Speaking to BFM television, she said an amendment proposed by the small LIOT faction in parliament and backed by left-wing parties would be declared "inadmissible".
She was alluding to Article 40 of the constitution, which bans legislative proposals from MPs that would add a burden to public finances.
Opponents of the pension reform had seen LIOT's parliamentary maneuver as their last hope of thwarting the changes, having previously tried and failed with an appeal to the country's constitutional court.
Reversing the increase in the retirement age, the key measure of Macron's hard-fought pension reform, would add billions to government spending, she said.
"You don't bend the constitution to please the opposition," government spokesman Olivier Veran said.
LIOT called the speaker's decision "an unprecedented attack on the rights of parliament."
Mathilde Panot, a senior figure from the hard-left France Unbowed (LFI) party, said that her group would submit a no-confidence motion and that discussions with partners were ongoing over the tactics to adopt.
"It's unacceptable for them to carry out such an act without there being a reaction," Panot said.
Senior Socialist party MP Boris Vallaud said different groups were debating the issue, while LIOT leader Bertrand Pancher said his MPs would decide later on Wednesday.
Observers said Prime Minister Elisabeth Borne's minority government risked losing a vote on the LIOT legislation, however, with left-wing parties, the far right and some centre-right MPs prepared to vote against the executive.
Panot said Braun-Pivet's decision was an admission of "fear" and accused her of failing to uphold the neutrality of her post.
The original legislation was rammed through the National Assembly in March without a direct vote using a controversial constitutional power that can be invoked by the prime minister.
The move led to accusations that Macron was riding roughshod over French democracy and public opinion, with around two-thirds of voters opposed to the changes, according to polls.
"The democratic cost will be larger than the economic cost they're hoping for," Vallaud said.