The incident happened on the M9 near Plean around one mile east of the Bannockburn Interchange at around 6.50am on Saturday.
Police said that a grey Toyota Hilux-type vehicle travelling eastbound left the road, coming to a stop in a field.
Two men were pronounced dead at the scene while a third man was taken to Forth Valley Royal Hospital with minor injuries.
The road was closed between junctions eight and nine for around 10 hours while police carried out investigations at the scene.
Officers are now appealing for witnesses and asked anyone with information to contact them.
Sergeant David Marr, of Police Scotland’s road policing unit based at Stirling, said: “Tragically, as a result of this collision two people have lost their lives and our thoughts are with their friends and families at this time.
“I would ask anyone who was on the M9 immediately prior to the collision, or who witnessed what happened, to get in touch as soon as possible.
“I also would appeal to anyone who may have dash cam footage of the incident to come forward to assist with our investigations.
“Similarly, anyone with other information relevant to the inquiry is asked to contact us as soon as possible.”
Those with information are asked to contact Police Scotland on 101, quoting incident number 902 of 17 March, or Crimestoppers on 0800 555 111 where anonymity can be maintained.