The Interstate passes through the Highland Rim and Nashville Basin physiographic regions of Tennessee, and is often used as the dividing line between the eastern and western portions of the former.

Since its completion, the rapid growth of the Nashville metropolitan area, as well as the general increase in traffic, has necessitated many widening and reconstruction projects.

Continuing through mostly rural territory characterized by slight rolling hills, I-65 gradually descends off of the Highland Rim into the Nashville Basin, and crosses the Elk River.

I-65 meanders over the next several miles through a series of irregular hills and valleys characteristic of the region's topography, and contains several minor curves along this stretch.

Bypassing Lewisburg to the west, I-65 passes through an artificial cut in a ridge, and traverses additional hilly terrain that is mostly wooded before straightening out.

After passing east of Spring Hill and the town of Thompson's Station, the freeway widens to six lanes and reaches a combination interchange with I-840, which serves as an outer southern bypass of Nashville.

I-65 has an interchange with the eastern terminus of SR 248 (Goose Creek Bypass) southeast of Franklin, one of the principal cities of the Nashville metropolitan area.

[5] Serving as the boundary between Oak Hill and the southern neighborhoods of Nashville, I-65 has an interchange with SR 255 (Harding Place) about three miles (4.8 km) later.

About one mile (1.6 km) later, I-40 splits off to the west, heading toward Memphis, and I-65 curves sharply to the northeast, reaching an interchange with US 41A (Rosa L. Parks Boulevard) immediately beyond.

[5] About one mile (1.6 km) later, I-65 crosses the Cumberland River on the Lyle H. Fulton Memorial Bridge and then reaches an interchange with I-24, beginning a concurrency with that route and shifting into a northward direction.

Slightly over one mile (1.6 km) later is a complicated stack interchange with US 31W/US 41 (Dickerson Pike) and SR 155 (Briley Parkway), the latter of which is a freeway that serves as a northern bypass around Nashville.

The widest section of highway in Tennessee is found on the north side of this interchange, where the road briefly accommodates a total of 15 lanes (eight northbound, seven southbound).

A little over three miles (4.8 km) later, the Interstate reaches an interchange with the western terminus of SR 386 (Vietnam Veterans Boulevard) in Goodlettsville, a freeway spur which serves the Nashville suburbs of Hendersonville and Gallatin.

[5] Leaving the urban Nashville area, I-65 reduces back to four lanes at SR 174 (Long Hollow Pike) and enters Sumner County.

Passing through rural terrain characterized mostly by farmland, I-65 expands back to six lanes and reaches an interchange with SR 25 about five miles (8.0 km) later near the town of Cross Plains, which also connects to Springfield and Gallatin.

[2] A planned interchange with US 431 in southern Williamson County was moved 4 miles (6.4 km) north to SR 248 (Peytonsville Road/Goose Creek Bypass) in October 1960 at the urging of the state highway department.

Both Ellington and the state denied these allegations, claiming the decision was made after the location was determined to be more suitable to relieve congestion in downtown Franklin.

[33][39] The last segment between the Alabama state line and Nashville, 9.7 miles (15.6 km) between US 31A near Cornersville and SR 373, was let to contract on September 11, 1964,[32] and opened on November 22, 1967.

[46][47] Work on the Lyle H. Fulton Memorial Bridge across the Cumberland River in Nashville began in June 1967,[48] and the section between I-40 and I-24, which was then I-265, opened on March 15, 1971, along with the adjacent stretch of I-40 to the west.

[50] The two-mile (3.2 km) segment between Berry Road, near the present location of the I-440 interchange, and the split with I-40 south of downtown Nashville was opened on October 25, 1972.

[51][52] The 14-mile (23 km) segment between US 31W in Millersville and SR 25 near Cross Plains was let to contract in April 1970,[53] and dedicated and opened by then-Governor Winfield Dunn on December 15, 1972.

[61] Widening of the adjacent seven-mile (11 km) segment extending south to SR 96 in Franklin from two to four lanes in each direction began in May 1996 and was completed in September 1997.

In addition, the local government had advocated for the designation to be changed in order to help alleviate congestion caused by motorists following I-65 through the city.

[72][73] In 2005, a project to widen the six miles (9.7 km) of I-65 from four to eight lanes between SR 96 and I-840 was moved from low to high priority upon the urging of local legislators and the city of Franklin.

[77] On August 15, 2014, a gasoline tanker truck crashed into the partially-rebuilt SR 248 overpass and exploded, killing the driver and necessitating the replacement of this bridge.

[79] Although the area along I-65 north of Nashville has not grown as fast as the suburbs to the south, this stretch of highway is part of a major north–south freight corridor between Atlanta and Chicago, and in addition to trucks, also receives a large volume of tourist traffic.

[80][81] As part of a project to construct a new interchange with an extension of the northern terminus of SR 109, the northernmost one mile (1.6 km) of I-65 in Tennessee was widened to six lanes.

[83] Preliminary engineering for the remaining 23-mile (37 km) segment, which extends to SR 174 in Goodlettsville, first received funding under the IMPROVE Act, passed by the Tennessee General Assembly in 2017.

This legislation increased the state's fuel taxes and vehicle registration fees for the purpose of funding a backlog of 962 needed transportation projects.

[48][91] In addition, a number of short sections, interchanges, and bridges along the Interstate are named in honor of state troopers and other law enforcement personnel killed in the line of duty, as well as local politicians and other prominent citizens.