As with most primary interstates ending in 5, it is a major crosscountry, north–south route, connecting between the Great Lakes and the Gulf of Mexico.

It also serves as one of the main north–south routes through Nashville, Tennessee; Louisville, Kentucky; and Indianapolis, Indiana, each a major metropolitan area in its respective state.

From I-10, I-65 runs west of downtown Mobile and through the northern suburbs of the city before turning northeasterly towards Montgomery.

Then it passes close to Columbia and crosses Saturn Parkway, which brings travelers to the town of Spring Hill.

The widest stretch of I-65 in its entirety is in Louisville at Kentucky Route 1065 (KY 1065, Outer Loop), where the main line is 14 lanes wide.

Prior to the project, the John F. Kennedy Memorial Bridge (completed in 1963) carried traffic in both directions.

The project also included reconstruction of the I-65/I-64/I-71 convergence interchange just south of the Kennedy Bridge, as well as renovating the older span to carry six lanes of southbound traffic.

At one time, the 65-mile (105 km) stretch of I-65 from Louisville to Elizabethtown was a toll road, called the Kentucky Turnpike.

On February 12, 2007, a bill passed the Kentucky Senate to rename I-65 in Jefferson County the Dr. Martin Luther King Jr.

Miles 0–9 were rebuilt, widened, and realigned from north of Sellersburg to the Ohio River during 2008–2010, giving great traffic relief to the fast-growing Indiana suburbs of Louisville.

In mid-March 2007, a six-mile (9.7 km) section of I-70 from the North Split to I-465 east of downtown was restricted to automobiles only for the "Super 70" project, a massive rebuild and expansion of that freeway.

[4] Trucks over 13 short tons (12 t) were forced to divert through I-65 if coming from the north and use the circular I-465 to the south to reconnect to I-70 eastbound.

North of Lafayette near Brookston, the road passes through the Meadow Lake Wind Farm for several miles, with the turbines and standards spaced out in order to avoid a collapse onto the highway.

[14][15] I-65 was not officially completed until December 19, 1985, when a section north of Birmingham opened, replacing a four-lane stretch of US 31 that had been designated as part of I-65 but did not meet interstate highway standards.