The Nashville, Chattanooga and St. Louis Railway (reporting mark NC&StL) was a railway company that operated in the U.S. states of Kentucky, Tennessee, Alabama, and Georgia. It began as the Nashville and Chattanooga Railroad, chartered in Nashville on December 11, 1845, built to 5 ft (1,524 mm) gauge and was the first railway to operate in the state of Tennessee. By the turn of the twentieth century, the NC&StL grew into one of the most important railway systems in the southern United States.

The Nashville & Chattanooga Railway, predecessor to the NC&StL Railway, was organized in 1848 by a group of prominent Nashville businessmen. The line's first president was Vernon K. Stevenson, who was connected to wealth from the Grundy and Bass families of Nashville and was a vigorous promotor of a line between Nashville and Chattanooga; he would serve for 16 years. The first locomotive in Nashville arrived in December 1850 on the steamboat Beauty along with 13 freight cars and one passenger car. The train made its first trip the following spring: 11 miles (18 km) to Antioch, Tennessee. It took nine years to complete the 150 miles (240 km) of line between Nashville and Chattanooga, made difficult by the steep elevations of the Highland Rim and Cumberland Plateau between them. The 2,228-foot (679 m) Cowan Tunnel near Cowan, Tennessee, was considered an engineering marvel of the time. Due to terrain difficulties, the rail line crossed into Alabama and Georgia for short distances. Towns sprang up during construction, including Tullahoma and Estill Springs.

During the Civil War, the rail line was strategic to both the Union and Confederate armies. The Tennessee campaigns of 1862 and 1863 saw Union troops force the Confederates from Nashville all the way to Chattanooga, down the line of the railroad. The tracks and bridges were repeatedly damaged and repaired, and the rolling stock was largely destroyed. At different times the trains carried supplies for both armies. In 1885, the railroad successfully defended itself before the Supreme Court in Nashville, C. & St. L. R. Co. v. United States from repaying postage payments for mail in 1861 that was not delivered because of the war.

After the war, the company purchased the Nashville and Northwestern Railroad and the Hickman and Obion Railroad to Hickman, Kentucky, to reach the Mississippi River. In 1873, it was reincorporated as the Nashville, Chattanooga and St. Louis Railway (NC&StL); the company's tracks never actually reached St. Louis, Missouri, in the north. In early 1877, the NC&StL bought the bankrupt Tennessee and Pacific Railroad from the state government and operated it as a connection to Lebanon, Tennessee.

The company also took full control of the Duck River Valley Narrow Gauge Railway in 1888, converting it to standard gauge the following year. It had already leased the line, which linked Columbia, Lewisburg, and Fayetteville, Tennessee from its owners in 1879, when they had difficulty completing the final stretch into Fayetteville.

The railroad's only heavy repair shops for locomotives and cars were located in Nashville, Tennessee. The first roundhouse and machine shop were built in 1850, which were expanded by Confederate troops during the Civil War. By 1888 the shops had become obsolete and inadequate, so they were moved to a larger tract of land two miles west, below Charlotte Avenue. The new shops featured a full-circle roundhouse and a dozen shop buildings served by two transfer tables.

The Louisville and Nashville Railroad, an aggressive competitor of the NC&StL, gained a controlling interest in 1880 through a hostile stock takeover that caused much rancor between the cities of Nashville and Louisville. However, the railroads continued to operate separately until finally merging in 1957. The company gave up steam operations in 1953. After the 1880 takeover, the NC&StL acquired branch lines in Kentucky and Alabama, and expanded from Nashville to Memphis. In 1890 the tracks reached Atlanta, Georgia, by leasing the state-owned Western and Atlantic Railroad.

In 1902, the L&N was acquired by the Atlantic Coast Line Railroad in a takeover similar to that of the NC&StL, but continued to operate as a separate company. In 1982, the L&N's corporate existence ended when it was merged into ACL's successor, the Seaboard System Railroad. After several other mergers, in 1986 the Seaboard System was renamed CSX Transportation, which continues to use the original NC&StL route between Nashville, Chattanooga, and Atlanta. Other portions of the system, such as the route to Hickman, have been abandoned.