The collision happened at an intersection on the Hume Freeway at Chiltern at 10.30am on Thursday.
All four people inside the sedan were killed.
The 30-year-old driver of the B-double truck suffered non-life threatening injuries and has been taken to a local hospital.
The cause is still under investigation and detectives from the Major Collision Investigation Unit are on their way to the scene, which is about three-and-a-half hours from Melbourne.
It's understood the truck was heading north on the Hume Freeway and the car attempted to turn left from Wenkes Road onto the freeway when the crash happened, according to Victoria Police Acting Inspector Joel Hughes.
"It's terrible, unfortunately in regional Victoria this year we've seen a number of lives lost in our roads and this is another tragic incident," Acting Insp Hughes told reporters.
There's been 193 deaths on Victorian roads this year, up from 157 at the same time in 2022.
Acting Insp Hughes said police were still working to identify the victims and contact family members.
Traffic has been diverted away from the area and it's not known when the roads will reopen.
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