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The collision happened around 10:46 local time (01:46 GMT) while the workers were spraying herbicide, said the Tobu Railway, the operator of the line.
When emergency services arrived at the scene three workers were trapped under the train, while one was on the platform, police said.
Train service has been suspended on the Tobu Nikko Line due to the accident, according to Tobu Railway.
A passenger on the train told Japanese public broadcaster NHK that they heard a "strange noise" before the train made an emergency stop.
He said passengers were stuck on the train for a while as staff announced that they were investigating the noise.
Fatal train accidents are rare in Japan, which relies on a sprawling railway network.
The train involved in Thursday's incident was a limited express train, according to NHK. These are the second-fastest trains in Japan, after the Shinkansen or bullet train.
Usually, there would be an employee stationed nearby to warn workers on the tracks of approaching trains - but it was unclear what the situation was like on Thursday morning when the accident happened, the Asahi Shimbun newspaper reported.
"The usual lookout system was operating as normal - when a train approaches, a lookout waves a flag and, once the driver acknowledges it, sounds the horn," a representative from Tobu Railway told the BBC.
"We are currently investigating why the accident occurred despite this."
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