Interstate 65 begins its journey in Mobile at its junction with Interstate 10. From I-10, I-65 goes west of downtown Mobile and through its suburbs before turning northeast toward Montgomery. From the state's capital, I-65 heads north, bypassing Prattville and Clanton before going through Greater Birmingham. As the combined interstates of I-20/59 converge on Interstate 65 in Birmingham, many wrecks, and accidents that have occurred that include the melting of support beams of overpasses after 18-wheelers crashed and burned on two separate occasions, and the numerous collisions that happen every year, has led this interchange to be nicknamed, "Malfunction Junction".
From Birmingham, I-65 continues north toward Cullman and Decatur. At Decatur, motorists can take Interstate 565 eastward toward Huntsville.
From Decatur, the interstate continues northward through Athens to the Tennessee state line.
In 2004, following the death of President Ronald Reagan, a lengthy segment of I-65 from Jefferson County to Limestone County was designated "The Ronald Reagan Memorial Highway". The sign designating the north end of the segment includes a statement from Reagan's speech at Point Mallard Aquatic Center in nearby Decatur on July 4, 1984.
Hyundai's new automotive plant in Montgomery is located just off I-65. It can be accessed using the Pintlala-Hope Hull exit (number 164).