Members of the RMT trade union working on Virgin Trains East Coast have begun a 24-hour strike.
The action is the latest in a long-running dispute over jobs.
The union said about 1,800 members, including train crew and some drivers, were taking part in the industrial action.
Workers are angry about threatened job cuts and disputes over working conditions and safety.
General secretary Mick Cash said: “RMT will not sit back while nearly 200 members’ jobs are under threat.”
Virgin Trains said it would be running a full timetable during the strike.
Virgin Trains East Coast has revealed plans to reorganise customer-facing roles which would “see a single person take responsibility for the customer experience”.
“This will have no impact on safety, and will result in a better experience for customers,” explained the operator.
However, the RMT union claims the measures would lead to job cuts and impact on working conditions.
RMT general secretary Mick Cash said: “We will also not tolerate the cavalier attitude to safety that is now on show as the company mobilises its scab army of managers.”
Virgin Trains East Coast operates services between London, north-east England and Scotland.