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.”
He said cheaper hydrogen production may be required to make it a viable fuel since the gas remains far more expensive than petrol and diesel.
However, building hydrogen plants close to wind farms to exploit surplus energy generated overnight could significantly reduce the cost.
Dr Todd said: “We need to make a major change to the energy system, with more power from renewable sources to the grid.”
Hydrogen-powered trains have been carrying passengers in Germany since 2018, while other research projects have been launched in England.
The Scottish Government has pledge to “decarbonise” passenger trains by 2035, five years ahead of the UK.
Its Transport Scotland agency is examining the merits of both battery and hydrogen power following ministers’ commitment to trial hybrid self-powered trains.
Hitachi has already offered to add batteries to one of its new class 385 electric trains which run ScotRail services on several lines between Edinburgh and Glasgow. It said they would have a 60-mile range.
A Transport Scotland spokesperson said: “In line with our commitment to decarbonise the railway by 2035, hydrogen is one of the energy sources we are exploring as an alternative to diesel.
“We are working with Scottish Enterprise and rail industry partners to see how this can be practically applied to a retired class 314 ScotRail train.
Scottish Enterprise head of high value manufacturing David Leven said: “This rail innovation project will provide opportunities for Scotland’s manufacturing companies as well as support the delivery of our national ambitions for more and better jobs and a net zero carbon economy.”