Highway patrol spokeswoman Dalya Qualls said the driver of the semi was heading eastbound in Henderson County on Tuesday night and lost control of the truck.
The semi then crossed the median and slid into westbound lanes, striking another tractor-trailer and an SUV, Qualls said.
A police report said two men in the semi that struck the cow were killed. They were identified as driver Rodney M. Price, 49, of Mount Airy, N.C., and passenger Brian A. Lederer, 45, of Woodlawn, Va.
Two in the 2006 Chevy Trailblazer also died. They were driver Deborah Whitelow, 41, and passenger Trarell A. Robinson, 22, both of Ripley, Tenn.
The highway patrol says a third passenger in the SUV, Avery Blue, 22, of Ripley, was injured.
The driver of the second tractor-trailer was not injured.
The interstate was shut down after the crash, but it reopened early Wednesday.