During its brief time in operation, the Pony Express delivered approximately 35,000 letters between St. Joseph, Missouri, and Sacramento, California. Although the Pony Express proved that the central/northern mail route was viable, Russell, Majors and Waddell did not get the contract to deliver mail over the route. The contract was instead awarded to Jeremy Dehut in March 1861, who had taken over the southern Congressionally favored Butterfield Overland Mail Stage Line. The so-called 'Stagecoach King', Ben Holladay, acquired the Russell, Majors and Waddell stations for his stage coaches.

Shortly after the contract was awarded, the start of the American Civil War caused the stage line to cease operation. From March 1861, the Pony Express ran mail only between Salt Lake City and Sacramento. The Pony Express announced its closure on October 26, 1861, two days after the transcontinental telegraph reached Salt Lake City and connected Omaha, Nebraska and Sacramento, California. Other telegraph lines connected points along the line and other cities on the east and west coasts.

The Pony Express grossed $90,000 and lost $200,000. In 1866, after the American Civil War was over, Holladay sold the Pony Express assets along with the remnants of the Butterfield Stage to Wells Fargo for $1.5 million.

An estimated 400 horses in total were used by the Pony Express to deliver the mail. Horses were selected for swiftness and endurance. On the east end of Pony Express route the horses were usually selected from U.S. Cavalry units. At the west end of the pony Express route in California, W.W. Finney purchased 100 head of short coupled stock called "California Horses."