U.S. Route 45 is a north–south United States highway. US 45 is a border-to-border route, from Lake Superior to the Gulf of Mexico. A sign at the highway's northern terminus notes the total distance as 1,300 miles (2,100 km).

US 45 is notable for incorporating, in its maiden alignment, the first paved road in the South, a 49-mile segment in Lee County, Mississippi. Let to contract in July 1914, the concrete highway opened on November 15, 1915.

As of 2006, the highway's northern terminus is in Ontonagon, Michigan, at the corner of Ontonagon and River Streets, a few blocks from Lake Superior. M-64 formerly terminated there as well until its rerouting in October 2006 to use the newly built Ontonagon River Bridge. Its southern terminus is in Mobile, Alabama, at an intersection with U.S. Route 98.

U.S. 45 is co-routed with unsigned State Route 17 between Mobile and Vinegar Bend, just north of Deer Park, in Washington County, Alabama. From Vinegar Bend to the Mississippi state line, U.S. 45 is paired with unsigned Alabama State Route 57.

U.S. Highway 45 is part of a designated hurricane evacuation route in Mississippi. It is entirely four-laned from its point of entry from Alabama, at the town of State Line, to the Tennessee line just north of Corinth, along the way serving the towns of (from south to north) Waynesboro, Meridian, Columbus and Tupelo.

At Brooksville, U.S. 45 splits away from U.S. 45 Alternate and serves the towns of Columbus and Aberdeen before rejoining U.S. 45 Alternate south of Tupelo. The alternate roadway provides a more direct and entirely four-laned route between Meridian and Tupelo, bypassing Columbus to the west and, more closely, Starkville to the east. The longer mainline route includes a segment of two-lane roadway immediately north of the split with 45-Alternate and continuing to US 82. Construction is presently underway to widen this entire segment, with final completion expected in September 2012.

Major junctions of U.S. 45 in Mississippi include U.S. Route 84 at Waynesboro, Interstate 20/59 at Meridian, U.S. Route 82 at Columbus, future Interstate 22 (present U.S. Route 78) at Tupelo and U.S. Route 72 at Corinth. Each of these junctions is an interchange and, with the exception of Waynesboro, each is part of a freeway segment.

The Mississippi section of U.S. 45 is defined at Mississippi Code Annotated § 65-3-3.

From the Mississippi state line U.S. 45 extends north past Selmer and Henderson and Jackson to Fairview, just north of Jackson. From there to the northwest, U.S. 45 and 45W are concurrent with unsigned Tennessee State Route 5 from the Mississippi state line to Union City and then with U.S. 51 (unsigned SR 3) to the Kentucky state line. From Fairview to the northeast, U.S. 45E is concurrent with unsigned Tennessee State Route 43 for most of the route's length, except for short segments at Martin and South Fulton where it is cosigned with Tennessee State Route 216 and Tennessee State Route 215 respectively. It rejoins U.S. 45 and 45W at South Fulton.

U.S. 45 enters Kentucky at Fulton then northeast past Mayfield then heads directly north into Paducah as a four-lane highway. In Paducah, U.S. 45 serves as a major artery, intersecting with Interstate 24 at exit 7, and intersecting US 60 and 62. U.S. 45 leaves Kentucky from Paducah's northern border across a two-lane, metal-grate bridge to Brookport, Illinois across the Ohio River.

In the state of Illinois, U.S. 45 runs from the bridge across the Ohio River from Paducah, Kentucky, through Shawnee National Forest and north to the Wisconsin border east of Antioch, Illinois. With a length of 428.99 miles (690.39 km) in Illinois, U.S. 45 is the longest numbered route in Illinois.

In its progress north from the Ohio River U.S. 45 first joins Interstate 24 as far as Vienna then heads northeast thru Harrisburg and north thru Fairfield, Flora, Effingham, Mattoon, Champaign, Urbana, Gilman and Kankakee, then straight north thru the western suburbs of Chicago in Will County, Cook County and Lake County to the Wisconsin border.

U.S. 45 enters the state in southeast Wisconsin. It runs concurrent with Interstate 894 and U.S. Route 41 through the west side of metro Milwaukee to form a major artery through the metropolitan area. It runs north to Fond du Lac. The highway routes near the west coast of Lake Winnebago through Oshkosh, Wisconsin. U.S. 45 then travels north through Appleton, Wittenberg and Eagle River, as well as the state and national forests, until it leaves the state at Land O' Lakes and enters Michigan.

US 45 enters Michigan south of Watersmeet. From there, the highway crosses the Western Upper Peninsula through the Ottawa National Forest running north to Ontonagon. US 45 ends just south of Lake Superior in downtown Ontonagon. The terminus was not changed in 2006 despite realignment then of M-38 and M-64 from the terminus to a crossing 0.7 miles (1.1 km) south.

Until 1934, US 45's northern terminus was in the Chicago, Illinois area.

Prior to the construction of the Interstate Highway system, US 45 was one of the main routes south out of Chicago toward New Orleans, LA. Much of the traffic left US 45 at Effingham, Illinois, continuing on through Cairo, Illinois along Illinois Route 37.