Did you make a change on your line?
I had a similar problem before, made an extension to my train line to incorporate a couple of extra cities and instead of going through the hassle of trying to fit them in in the right order I deleted ALL stops and started again – I can only assume that therefore my line ceased to exist (as far as the ‘sims’ were concerned) and they all decided to walk to their destinations instead.
This sent me broke for a time, from being a couple of million up to half a million in the red, my trains were costing me a fortune in maintenance but not carrying any passengers for a couple of years – eventually everything got back to normal thankfully.