Pendleton was the son of Nathanael Greene Pendleton and attended the local schools and Cincinnati College and the University of Heidelberg in Germany. Pendelton studied law and was admitted to the bar in 1847 and commenced practice in Cincinnati.

He was a member of the Ohio Senate from 1854 to 1856. In 1854 he ran unsuccessfully for the Thirty-fourth United States Congress. Three years later he was elected as a Democrat to the Thirty-fifth Congress and also succeeded in being reelected to the three following Congresses (March 4, 1857 to March 3, 1865), but in 1864 he failed to be elected to the Thirty-ninth Congress.

He was one of the managers appointed by the House of Representatives in 1862 to conduct the impeachment proceedings against West H. Humphreys, United States judge for several districts of Tennessee. He ran in the 1864 U.S. presidential elections for Vice President, together with George McClellan. Their opponents were Abraham Lincoln (President) and Andrew Johnson (nominee for Vice President).

Pendleton also failed to be elected to the Fortieth Congress and was an unsuccessful Democratic candidate for Governor of Ohio in 1869.

In 1869 he became president of the Kentucky Central Railroad and kept this position until he was elected as a Democrat to the United States Senate in 1879. He served six years in the Senate from March 4, 1879, to March 3, 1885 but was unsuccessful for renomination. During this time, he sponsored the Pendleton Act of 1883 in response to the assassination of President James A. Garfield by Charles Guiteau. The Act helped put an end to the system of patronage that was in widespread use at the time.

From 1881 to 1885 he was Chairman of the Democratic Conference. He was appointed Envoy Extraordinary and Minister Plenipotentiary to Germany in 1885, and served until his death in Brussels, Belgium. He is interred in Spring Grove Cemetery, Cincinnati, Ohio.

The city of Pendleton, Oregon is named after him.