Workers on Northern Rail - which runs train services across South Yorkshire - are going to be balloted for strike action.
Transport union RMT said they are taking action in a row over the role of guards.
Union bosses have informed Arriva Rail North that conductors who are union members are now in formal dispute and will begin immediate preparations for a ballot for industrial action.
The RMT said this comes after the firm failed to provide assurances over the future of the safety critical role of the guard during a recent meeting.
RMT general secretary Mick Cash said: "The union’s position on 'driver only operation' is perfectly clear. We will not agree to any extensions of DOO and will fight to retain the safety critical role of the guard and to keep a guard on the train.
“We asked Arriva Rail North whether they were prepared to guarantee a second person on all passenger services operated by the company and whether that second person would retain full operational responsibility, for train dispatch, platform train interface and current rule book requirements for dealing with contingency arrangements, such as evacuation and protection of the train.
“The response from the company was ‘we are not in a position to offer either of these guarantees at this present time’."
A spokesman for Northern Rail called the ballot for strike action 'extremely premature.'
He added: "During our discussions this week we offered commitments to consult fully with our people, customers and key stakeholders. We want to protect jobs and pay as we work together to provide safe, secure and accessible services."