Part of the Hume Fwy was closed early on Monday, March 20, after two trucks collided at Balmattum.
Police said the trucks were believed to have collided just after 1.30am.
A man driving one of the trucks was taken to hospital with non-life-threatening injuries, police said.
The driver of the second truck was not injured.
The south-bound section of the road was closed between Violet Town and Euroa for several hours while police undertook investigations, which are ongoing.
The road reopened shortly before 8am.
Anyone who witnessed the collision or has dashcam footage is urged to contact Crime Stoppers on 1800 333 000 or submit a report online at www.crimestoppersvic.com.au