The route passes through the major cities of Montgomery, Birmingham, Decatur, Huntsville, and Athens before entering Tennessee in the north near the town of Ardmore, Alabama.

The entire Alabama portion of I-65 is dedicated as Heroes Highway in honor of Central Intelligence Agency officer Johnny Micheal Spann and all of the people who died during the September 11 attacks.

As the I-65 approaches the Montgomery city limits, the Hyundai Motor Company's automotive plant can be found just off freeway.

Approximately halfway between Montgomery and Birmingham, it passes Clanton, the county seat, where the water tower, visible from the road, is shaped and painted to resemble a huge peach.

As the Interstate passes by downtown Birmingham, southbound travelers have a view of the Vulcan statue atop Red Mountain.

At the north edge of downtown, I-65 reaches the I-20/I-59 intersection (exit 261) with a crossover interchange, often called "Malfunction Junction", through which the highway has five lanes, continuing on northward with four.

It is here that I-65 meets the eastern terminus of I-22, which heads northwest to Memphis, Tennessee, filling in a gap in the Interstate Highway System.

The Interstate then continues 98 miles (158 km) in the general direction of Huntsville, crossing the Blount–Cullman county line (milemarker 291), where lanes transition from three to two, as well as passing the city of Cullman on the way.

The highway connects the Huntsville–Decatur metropolitan area as it crosses Wheeler Lake (Tennessee River) on a 2.6-mile (4.2 km) bridge.

Between Walkers Chapel Road in Fultondale and the Tennessee River in Decatur (Limestone County), I-65 has been designated the "Ronald Reagan Memorial Highway".

The unique feature of this rest area compared to others is the large Saturn IB rocket erected on the site as a memorial to Alabama's—and, in particular, Huntsville's—contribution to NASA's space exploration.

[citation needed] The first section of Interstate in Alabama opened to traffic was the eight-mile (13 km) stretch of I-65 between northern Jefferson County and Warrior on December 10, 1959.

These accidents include two separate occasions of the support beams melting after crashes by 18-wheelers and the numerous collisions that happen every year, resulting from the junction with I-20 and I-59.

The sign designating the north end of the segment includes a statement from Reagan's speech at Point Mallard Park in nearby Decatur on July 4, 1984.