The Liberation Tigers of Tamil Eelam (LTTE) set off the blast in the district of Vavuniya, 250 kilometres (150 miles) north of Colombo just before midnight on Monday, the ministry said.
Military officials said the blast was caused by a mine planted by the side of the road to target the vehicle.
It came two hours ahead of a rebel air attack against government troops in the Jaffna peninsula, further north of Vavuniya, officials said.
The rebels said they staged the air attack using two of their light aircraft.
Earlier this month the Tigers were blamed for a blast that hit another bus in the same area of Vavuniya, killing eight passengers and wounding 25.
That attack came five days after a bomb exploded inside a bus in the east of the island, killing 17 people and wounding 25.
More than 4,000 people have been killed in the latest upsurge of violence that began in December 2005, despite a truce arranged in 2002 to end the bloody ethnic conflict.
The Tigers have wag