Govia Thameslink Railway is taking a legal case against union Aslef to the Supreme Court over its industrial action on Southern Railway.
The move came on the second day of strike action by the union in a bitter dispute over driver only trains.
A further strike will be held on Friday as well as three more walkouts at the end of the month.
The strikes have halted most of Southern's 2,200 daily services. GTR lost a court case and an appeal last year to try to stop the strikes.
A statement said: "GTR is determined to protect its passengers and its business from unlawful industrial action.
"GTR is therefore prepared to continue its legal claim to the Supreme Court, as it believes that it has an arguable case that the industrial action is unlawful under EU law.”
Last month, the High Court rejected an argument from GTR that industrial action would breach customers' rights.
Aslef described last month's legal action as a waste of taxpayers, shareholders and passengers' money.
There was no immediate comment from the union to the new legal move.
Aslef members are continuing to ban overtime, which has led to delays and cancellations.