I-65 passes through three of Kentucky's ten largest cities—Bowling Green, Elizabethtown, and Louisville—and serves Mammoth Cave National Park and Fort Knox.

I-65 also has interchanges with I-165 (formerly the William H. Natcher Parkway) near Bowling Green, I-265, I-264, and a complex junction with I-64 and I-71 along the south bank of the Ohio River in central Louisville.

[5] On December 30, 2016, both I-65 bridges began using electronic toll collection (ETC) to charge motorists for their use of this previously toll-free Interstate crossing.

Unlike most states, Kentucky law requires that tolls be removed when the original construction bonds are paid off and cannot be extended.

In addition to toll plazas, the Kentucky Turnpike also provided two service areas just south of Lebanon Junction and just north of Shepherdsville.

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

[14] On July 15, 2007, Kentucky officially raised its speed limits on Interstate and state parkway highways to 70 mph (110 km/h).

[citation needed] In July 2017, the KYTC opened a new interchange of I-65 at milemarker 30 to provide access to the Kentucky Transpark near Bowling Green.

The first phase of the project include the new interchange, exit 30, plus a four-lane connector road going from the Interstate to US 68 just east of Bowling Green.

[17] The second phase is building a two-lane connector road running from US 68 to US 31W between Bowling Green and Oakland, thus relieving congestion problems on both U.S.