In Wyoming, the Interstate Highway runs 300.530 miles (483.656 km) from the Colorado state line near Cheyenne north to its national terminus at I-90 near Buffalo.

The highway also has extensive concurrencies with US 20 and US 26 along its east–west segment through the North Platte River valley.

The Interstate has business loops through Cheyenne, Chugwater, Wheatland, Douglas, Glenrock, Casper, and Buffalo.

The four-lane Interstate has its first interchange, a diamond interchange, with Wyoming Highway 223 (WYO 223, Terry Ranch Road), and crosses over the Union Pacific Railroad's Speer Subdivision rail line and over the BNSF rail line.

The freeway has a dumbbell interchange with Vandehei Street before leaving the city of Cheyenne and passing through the unincorporated area of Ranchettes.

The Interstate enters Platte County at its interchange with Bear Creek Road.

The Interstate meets US 26 at a directional T interchange at Dwyer Junction, and the U.S. Route joins I-25 and US 87 heading north.

The Interstate has an interchange with Wyoming Highway 319 Spur (WYO 319 Spur) in the town of Glendo, then parallels WYO 319 and the BNSF rail line northwest into Converse County to the west of the Glendo Reservoir, an impoundment of the North Platte River.

I-25 crosses over the BNSF rail line and the North Platte River, then curves northwest to meet the business routes at a trumpet interchange on the west side of the city near the business routes' intersection with WYO 59, which leads north to Gillette.

West of the river, the freeway has a diamond interchange with WYO 220 (Poplar Street), then curves north and has a diamond interchange with US 20 and US 26, which split west toward Shoshoni and Casper–Natrona County International Airport, and the access road to the Casper Events Center.

[1] I-25 leaves the city of Casper and has an interchange with WYO 254 (Wardwell Road) next to unincorporated Hartrandt and the town of Bar Nunn.

US 87 continues north along westbound I-90 toward Sheridan and Billings; I-90 also heads east toward Gillette.

[2] Earlier funds from the state government had been used to begin work on a freeway bypass of Casper in 1953; its first 3-mile (4.8 km) section was opened to traffic on October 1, 1960.

[3] The final section of the freeway, spanning 26 miles (42 km) between Casper and Kaycee, was opened on February 2, 1982.

Its business loops are located in Cheyenne, Chugwater, Wheatland, Douglas, Glenrock, and Casper.