Angela Rayner has tabled a motion to block Sadiq Khan from returning to Parliament as an MP as insiders have pointed to her "making manoeuvres" towards leadership.
New legislation introduced by the Deputy Prime Minister would prohibit elected mayors from simultaneously holding parliamentary seats, effectively preventing the London Mayor from returning to Westminster.
The English Devolution Bill proposes amendments to the Greater London Authority Act 1999, stipulating that individuals cannot occupy mayoral positions if they serve as "an elected member of a legislature in the United Kingdom".
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It is understood the restriction would extend beyond London, affecting all city mayors across England.
Insiders suggest the Deputy Prime Minister is "on manoeuvres" to obstruct potential Labour leadership rivals.
One Conservative strategist told the Telegraph: "Fresh out of trying to do in Wes Streeting, yet again Angela Rayner is on manoeuvres, knifing potential leadership rivals."
They added: "She should focus on delivering for Britain rather than on her petty party political squabbles. Yet again, it's party first, country last."
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Mrs Rayner's plans would prohibit elected mayors from simultaneously holding parliamentary seats
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Mr Khan previously represented Tooting as a Labour MP from 2005 until assuming the mayoralty in 2016.
Defending the legislation, the Ministry of Housing, Communities and Local Government said it would create uniformity across mayoral positions.
A departmental spokesman explained: "It is already the case that mayors who are police and crime commissioners cannot also serve as MPs, and these changes will make this consistent for all mayors so that they can focus on what is a significant role at the forefront of delivering change."
Despite Mrs Rayner's moves, Mr Khan has repeatedly dismissed suggestions of leadership ambitions.
Anegla Rayner's move has been seen as 'knifing potential leadership rivals'
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Last year, he said he had "no interest at all to be a Labour leader or the next Labour leader" and "no ambitions to go back to the Commons".
Currently serving his third mayoral term following re-election in May, Mr Khan is widely expected to step down when this term concludes.
Rayner has consistently denied harbouring leadership ambitions, telling Sky News: "Absolutely not. I don't want to be leader of the Labour Party."
A spokesman for Sir Sadiq declined to comment.
Earlier this month, Dominic Cummings gave his take on Mrs Rayner's chances of taking over from Sir Keir Starmer
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Speaking exclusively to GB News earlier this month, Dominic Cummings gave his prediction on Mrs Rayner's chances of taking over from Sir Keir Starmer as Labour leader.
He told GB News' Steven Edgington: "The Labour Party doesn't get rid of people. And these kinds of heuristics are quite sticky historically.
"But I now think as this old system really crumbles, maybe these old rules of thumb become less important.
"It strikes me as more and more that [Starmer's] removed and that Rayner takes over. He can't do the job. He doesn't understand the job. He doesn't like the job."