Cullom was born in 1829 in Monticello, Kentucky. He moved with his family a year later to Tazewell County, Illinois. During his youth, Cullom assisted his father with farm labor. Cullom attended the Mount Morris Seminary for two years and became a Teacher. Under influence from his father's Whig beliefs, Cullom became interested in politics. He moved to Springfield, Illinois in 1853, where he studied law with Stuart & Edwards and was admitted to the bar in 1855. He practiced law in Springfield with Charles S. Zane, and was elected city attorney in 1855.

Cullom was elected to the Illinois House of Representatives as a Whig in 1856, serving one term. With the disintegration of the Whig party, Cullom identified with both the Republican and the American parties. He was a candidate for elector on the American party ticket during the 1856 election. In 1860, he was re-elected to the Illinois House as a Republican, serving as Speaker in 1861.

In 1863 he married Julia Fisher, of Springfield, Illinois, who helped Senator Cullom in his political career.

He was elected in 1864 to the Thirty-ninth, and reelected to the Fortieth and Forty-first Congresses (March 4, 1865 – March 3, 1871). Cullom returned to the Illinois House from 1873 to 1874, serving again as Speaker. In 1876, he was elected Governor of Illinois, defeating Lewis Steward by 6,834 votes. He was re-elected in 1880, becoming the first Illinois governor to be re-elected after a four-year term. Under Cullom's governorship, the Southern Illinois Penitentiary was commissioned, the Great Railroad Strike of 1877 was quelled, the Illinois Appellate Court was founded, and the Illinois State Board of Health was established. He resigned in 1883 to take office as a US senator; Lieutenant Governor John Marshall Hamilton assumed the governorship in his place. Cullom was elected to the United States Senate in 1882, and reelected in 1888, 1894, 1900 and 1906, serving from March 4, 1883 to March 3, 1913.

As a Senator, Cullom oversaw the passage of the Interstate Commerce Act of 1887. He believed that only the federal government had the power to force railroads to provide fair treatment to all of its customers, large and small. This was because corporations, such as Standard Oil, had corrupted many of the railroad's officials into providing them with rebates, and as a whole, the companies in question were more powerful than any state government.

Cullum had an interest in the territories of the United States of the time. Together with Congressman Isaac S. Struble, Cullom pushed the Cullom-Struble Bill, whose sanctions against polygamy included exclusion of the Utah Territory from statehood. The bill was on the verge of passing Congress in 1890, but the legislation was preempted when The Church of Jesus Christ of Latter-day Saints (LDS Church) formally disavowed polygamous marriages with the 1890 Manifesto. Cullom was appointed by President william McKinley in July 1898 to the commission created by the Newlands Resolution to establish government in the Territory of Hawaii.

He died in 1914 in Washington, D.C. and is buried in Oak Ridge Cemetery in Springfield. Cullom was a close personal friend and associate of Jacob Bunn and John Whitfield Bunn, the Illinois industrialist brothers who contributed to the building of hundreds of millions of dollars of Business enterprises by 1900. The village of Cullom, Illinois is named in his honor.