The experimental train will be created by converting ScotRail carriages retired in December.
It is likely to be tested on a heritage line in Scotland like the Bo’ness and Kinneil Railway, which has been used for trials of battery trains.
Testing would then be switched to a main line.
Conversion work will be done at train refurbisher Brodie Engineering in Kilmarnock. The technology involved will be developed by London-based Arcola Energy, with the University of St Andrews also involved in the Scottish Enterprise project.
Arcola chief executive Dr Ben Todd said hydrogen could be used on non-electrified routes which were too long for battery-powered trains.
He said: “It will be a small feasibility study, involving the refit of a former ScotRail class 314 electric train.”