The Florida Highway Patrol said the tractor-trailer ran through a stop sign around 6:30 a.m. and struck the van's right side.
Both vehicles rolled over and landed on the shoulder of State Road 29, a two-lane highway about 60 miles east of Fort Myers.
At least two of the six men inside the 1993 Chevrolet van were thrown from the vehicle. All six were pronounced dead at the scene.
Officials have not yet released their names, pending notification of their families. Some of the individuals, all of them adults, were not carrying any identification.
Ewing Saunders, 66, was driving the tractor-trailer for West Coast Aggregate Haulers, Inc., of Venice, Fla., authorities said.
Saunders was taken to a hospital in serious condition. His injuries were not believed to be life threatening, Lt. Christopher Miller said.
Miller said charges were pending completion of the investigation.
West Coast Aggregate Haulers declined to comment on the crash.