The 43-year-old ticket examiner required hospital treatment after an incident on the Largs to Glasgow Central train as it approached West Kilbride station just after 3pm yesterday.
Paramedics also treated a 40-year-old man at the scene for a minor injury.
Two men and a woman were arrested and appeared at Kilmarnock Sheriff Court today in connection with the incident.
James Bunce, 35, from Largs, was charged with assault along with Thomas McCulloch, 21, from Saltcoats, and Terri Reid, 26, from Ardrossan.
They made no plea or declaration and will appear in court again at a later date.
A ScotRail spokeswoman said: “We do not tolerate crime on the railway and we are fully co-operating with British Transport Police in their investigation.”
The train worker is being treated at Crosshouse Hospital where his condition is described as stable.