A rail operator had been warned of cows on the track almost an hour before a train hit two of the stray cattle and derailed.
A driver reported seeing a cow on the line between Wye and Chilham in Kent at 8.50pm, Network Rail said.
The sighting was investigated, but nothing was found before the collision at around 9.40pm.
Sixty-five passengers who were on board at the time were evacuated by emergency services, and no one was injured.
Two carriages were derailed but remained upright, Kent Fire and Rescue Service said.
A Network Rail spokesman said: "At 8.50pm on Sunday we received a report of a stray cow close to the tracks between Wye and Chilham.
"The sighting was investigated by Network Rail staff in the area as well as two trains which passed through the area. Neither our people on the ground nor the train drivers reported any cows on the line."
He added that the fences in the location were last inspected three weeks ago and found to be in good condition.
Passenger Jonathan Calder tweeted a picture from the train which was derailed near Olantigh Road, showing the carriage in front of his at an angle, with the right-hand side of it clearly no longer on the rails.
He wrote: "Everyone is fine but this train is going nowhere. Front coach derailed."
A Southeastern spokesman said: "We would like to thank our driver who acted quickly after his train collided with two cows on the track. He realised at this point his radio had stopped working so he jumped out of the cab and ran down the track to halt an approaching train.
"He was then able to contact the signaller through the radio of this train to switch off the power to the lines.
"We can confirm no one was injured in this incident and all 65 passengers on board were taken from the train to a nearby village hall where they were given refreshments and buses and taxis were arranged to take them home."
Trains between Ashford and Ramsgate via Canterbury West have been suspended today and replacement buses have been put on while work is done to remove the train from the track.