Emergency services responded to reports of the collision, south of Heathcote, just before midday.
The driver of the ute, who is yet to be formally identified, died at the scene.
The driver of the truck was airlifted to Royal Melbourne Hospital in a stable, non-life threatening condition.
Victoria Police Eastern Division Region 3 Inspector Michael Layton said police had launched an investigation.
“We are in the very early stages,” Insp Layton said.
“We don’t know what the cause is, but we have the Heavy Vehicle Unit and Highway Patrol Unit working through that process at the moment.
“The vehicles were travelling in opposite directions and the impact is quite extensive.”
Due to the nature of the collision, a Hazmat team was required to deal with spilled chemicals at the scene.
The Northern Highway was closed for many hours on Wednesday.
Southbound traffic was diverted onto the Nagambie-Heathcote Road and northbound traffic was diverted onto the Seymour-Tooborac Road.
Insp Layton issued a reminder for motorists to be alert.
“We urge people to take care and consideration around driving habits,” he said.
“This area, the Mitchell area, has a high incidence of collisions on arterial roads.
“We say to people to drive appropriately to the conditions and monitor fatigue.”
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