Labour MP Ruth Smeeth has resigned from the party's front bench "in order to vote against a second referendum".
Labour had called on its MPs to abstain on an amendment calling for a second referendum.
The MP announced she would be stepping down as voting was under way in the Commons this evening (14 March), saying she had a duty to "support the will of my constituents" in Leave-supporting Stoke-on-Trent North.
Nearly 70% of voters in Stoke-on-Trent voted to leave the European Union in 2016.
Three more senior frontbenchers also rebelled to oppose a second referendum, even though Labour's official policy is to keep the option on the table.
The party's MPs had been told to abstain, but a total of 24 backed the call to delay Brexit in order to hold a second referendum, with 17 opposing it.