MP Dan Jarvis has been told he must stand down from parliament if he is elected as Sheffield City Region mayor.
Labour General Secretary Iain McNicol and incoming General Secretary Jennie Formby outlined the party's position in a letter to the Barnsley MP.
The party's National Executive Committee ruled last week that no-one could serve in two elected roles.
Mr Jarvis was given until 17:00 BST on Tuesday to make his position clear. The BBC has approached the MP for comment.
BBC political correspondent Iain Watson said that if Mr Jarvis refuses to adhere to the ruling Labour will discuss what action to take.
It is likely he would be removed as an MP if he is elected mayor on 3 May, Watson said.
He said Mr Jarvis insists he was selected as Labour candidate in the full knowledge he was proposing to do both jobs.
His supporters have suggested the party leadership is acting vindictively.
The Labour Party has previously said the ruling would only apply if Mr Jarvis was elected as mayor.
Analysis by James Vincent, BBC Look North political editor
Supporters of Dan Jarvis claim this is a last ditch attempt to stop him becoming Mayor. Or is it a last ditch attempt to stop him being Barnsley MP?
Labour says this is all about the issue of MPs having second jobs. According to its rules, he can't be an MP and mayor at the same time. However, it passed that rule two days before the nomination process ended. This 17:00 deadline comes the day before nominations open.
Labour could now decide to find another mayoral candidate. But it has got until next Friday and the party would have to find a person prepared to be unpopular in Yorkshire. Thirteen MPs from the county met on Tuesday to discuss the issue and all of them backed Dan Jarvis.