The accident happened around 7:30 am (2230 GMT) inside the 860-metre (940-yard) Hachihonmatsu tunnel in Hiroshima prefecture, southwest Japan, according to a local police spokesman who spoke with AFP on condition of anonymity.
A truck crashed into several vehicles that were stopped inside the tunnel, causing it and at least one other vehicle to catch fire, he said.
Local media said a dozen vehicles were involved, with at least five cars burning.
Emergency crews were seen assisting those who fled the tunnel, with a tent erected to assist those in medical need.
The fire was put out a little more than two hours after the accident, the police spokesman said.
Two people, including a woman, were confirmed dead, though the gender of the other fatality could not immediately be determined, the spokesman added.
Local media said about 70 other people were sent to hospitals for treatment and observation.
It follows a 2012 highway tunnel accident in central Japan where nine people were killed when concrete ceiling panels crashed onto three vehicles, setting at least one ablaze inside the Sasago tunnel, 80 kilometres west of Tokyo.
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