Sir Keir Starmer says he sacked his transport spokesman Sam Tarry for doing interviews “without permission” and “making up policy on the hoof”.
Left-wing supporters of Mr Tarry say the Labour leader fired him for joining striking rail workers on a picket line.
Mr Tarry himself says he was expressing “solidarity” with workers.
The BBC understands Sir Keir objected to an interview Mr Tarry gave to Sky News in which he said workers should get pay rises in line with inflation.
Labour’s policy is that there should be “fair” rises through negotiation.
Mr Tarry’s allies say many Labour-supporting unions back him and have expressed concern at his sacking.
Sharon Graham, leader of the Unite Union, which has more than a million members, said Labour was “becoming more and more irrelevant to ordinary working people”.
And speaking to the BBC, veteran Labour left-winger Diane Abbott pointed out that, unlike Mr Tarry, shadow chancellor Rachel Reeves had not been sacked for “making up policy on the hoof”.
Earlier this week, Ms Reeves gave an interview in which she said the party no longer backed renationalising the railways. Labour later clarified that renationalisation remained its policy.
Ms Abbott argued that Mr Tarry was being unfairly treated, adding: “Everybody knows Sam Tarry wasn’t sacked for interviews, he was sacked for going on a picket line.
“We are the Labour Party, the clue is in the name, we were set up by the trade unions – to penalise people, shadow ministers, for actually showing solidarity with the trade unions is wrong.”
Speaking to GB News, Greater Manchester Mayor Andy Burnham warned that Labour needed to be careful it didn’t come over as “a party that undermines working people fighting to protect their incomes and a cost of living crisis”.
On Wednesday, Mr Tarry gave media interviews from a picket line at London Euston station, in which he said he was “here as a shadow transport minister backing transport workers who are on strike”.
The Ilford South MP also told Sky News: “Every worker deserves a pay rise that is in line with inflation”.
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Talking to reporters in Birmingham, Sir Keir said he “completely” understood the “frustration of so many working people who’ve seen prices go up, seen inflation through the roof, and their wages haven’t gone up”.
He added that Labour would “always be on the side of working people”, but shadow ministers needed to maintain “collective responsibility”.
Asked if he was worried about losing trade union funding, Sir Keir said Labour had strong union links and had been working with them on writing employment rights legislation.
Responding to his dismissal, Mr Tarry said it was “a real shame” and argued the “vast majority” of people would be supportive of workers going on strike.
