The construction workers were in an active work zone on I-83 South at mile marker 35.5 in Fairview Township at the time of the crash, which is about 8 miles south of Harrisburg.
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A preliminary investigation determined that a 24-year-old man was driving a large box truck southbound on the interstate around 3:25 a.m. Wednesday when he collided with a construction vehicle in the active work zone.
The crash caused the box truck to continue onto the shoulder of the roadway, where it struck the three workers.
All three workers were pronounced dead at the scene.
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State police have not disclosed the victims' names, but Pennsylvania Department of Transportation spokesman Dave Thompson said they worked for an agency contractor.
"Right now we're just gathering information," Thompson said. "We're obviously very shaken by this event."
The crew was sealing highway cracks at the time, Thompson said.
A Transportation Department news release issued a week ago said the overnight work involved lane closures and was being performed by CriLon Corp. of Somerset. A phone message seeking comment was left early Wednesday at CriLon offices.
There has been no word on if the box truck driver will face any charges.
The Associated Press contributed to this post.