La Follette was born in a log cabin in the Town of Primrose, Wisconsin, just outside New Glarus, to Josiah La Follette and Mary Ferguson (widow of Alexander Buchanan). His paternal great-grandfather, Joseph La Follette, was born in France, emigrated to New Jersey, fought in the American Revolutionary War, led his family through the Cumberland Gap to Kentucky, and crossed the Ohio River into Indiana with his son, Jesse LaFollette. Joseph married Phoebe Gobel, whose family came to the Massachusetts Colony from England in the 1630s. Jesse's sons, Josiah and Harvey La Follette, moved to Primrose, where they established farms and participated in local government.

La Follette grew up in the village of Argyle, Wisconsin. The death of his father in 1856 and the subsequent bad relationship with his stepfather made it a difficult childhood. Following the death of his stepfather, his mother sold the family farm and moved to nearby Madison. He began teaching school for tuition money for the University of Wisconsin–Madison, where he was "a very mediocre student who enjoyed social activities."

At the school, he was deeply influenced by University President John Bascom on issues of morality, ethics and social justice. La Follette studied oratory and, during his senior year, won a major Midwestern oratorical competition. He graduated in 1879.

Soon after obtaining his law license, he gained the Republican nomination for Dane County District Attorney and won the seat in 1880. After two terms, he was elected to the United States House of Representatives, where he served three terms. There he was noted for championing Native and African-American rights. His opposition to patronage and his support for a protective tariff helped secure his appointment to the Ways and Means Committee headed by william McKinley, where he helped draft the Tariff Act of 1890 (McKinley Tariff).

La Follette met Belle Case while attending the University of Wisconsin, and they married on December 31, 1881, at her family home in Baraboo, Wisconsin. She became a leader in the feminist movement, an advocate of women's suffrage and an important influence on the development of La Follette's ideas. La Follette attended law school briefly and passed the bar in 1880. During this time, he became a vegetarian, declaring that his diet gave him more Energy and a clear head.

Born and raised in Wisconsin, he obtained a law license and won election as the Dane County District Attorney. In 1884, he was elected to the United States House of Representatives, losing his seat in the 1890 Democratic wave election. La Follette returned to Wisconsin to build up his law practice but remained active in politics, seeking the Republican nomination for governor in 1896, 1898, and 1900. He won the nomination in 1900, defeated his Democratic opponent in the general election, and served as Wisconsin's governor from 1901 to 1906. He sought numerous progressive reforms as governor, including workers' compensation and women's suffrage. While serving as governor, he won election to the United States Senate, holding office from 1906 to 1925.

After his defeat for a fourth term in the House, La Follette returned to Madison to begin a private law practice. In the early 1890s, he began to believe that much of the Republican Party had abandoned the ideals of its antislavery origins and become a tool for corporate interests. In his home state, he was convinced industry and railroad interests had too much sway over the party. To counter this, La Follette began building an independent organization within the party that stressed voter control.

In 1891, U.S. Senator Philetus Sawyer allegedly offered La Follette a bribe to side with him in a court case against former state officials. La Follette was outraged by Sawyer's proposal; the experience profoundly affected his nascent political career as, by his own lights: “Nothing else ever came into my life that exerted such a powerful influence upon me as that affair. It was the turning point, in a way, of my career. Sooner or later I probably would have done what I did in Wisconsin. But it would have been later. It would have been a matter of much slower evolution. But it shocked me into a complete realization of the extremes to which this power that Sawyer represented would go to secure the results it was after.”

La Follette spent the next six years creating a Republican bloc known as "The Insurgents" "in support of other party members (Scandinavians, dairy farmers, young men, disgruntled politicians) with grievances against the stalwart faction.” The Insurgents championed consumer rights and stressed the need for more direct voting including direct election of nominees in party primaries. Their calls for reform gained more support after the Panic of 1893 caused great upheaval in the economic and social conditions of most Americans.

In 1894, the Insurgents openly challenged the Stalwarts for leadership of the Republican Party. Their Nils Haugen sought the Republican nomination for governor in 1894, and La Follette followed in 1896 and 1898. His speeches decrying the sway of big Business (especially the railroads) and his calls for a more direct democracy drew ever increasing crowds.

In 1900, La Follette formed a coalition that temporarily disrupted the Stalwart hold on the nomination process. After securing the nomination, he "traveled to sixty-one counties, gave 216 speeches and spoke to 200,000 people." He gave many of his campaign speeches (which often lasted over three hours) from the back of a buckboard wagon. He won the 1900 race for governor by 100,000 votes and won re-election in 1904.

From 1901 until 1906, La Follette served as Governor of Wisconsin — the first native-born governor of the state. During his first term, he proposed to set up a railroad commission, imposed an ad valorem tax on the railroad companies, and established a direct primary system. The Stalwarts blocked his agenda, and he refused to compromise with them.

During the 1904 elections, the Stalwarts organized to oppose La Follette's nomination and moved to block any reform legislation. La Follette began working to unite insurgent Democrats to form a broad coalition. He did manage to secure the passage of the primary bill and some revision to the railroad tax structure.

In 1906, when La Follette went back to Washington, D.C., the American economy had changed due to an increasing number of mergers that consolidated financial power in fewer hands. Senators Nelson Aldrich and John C. Spooner were widely seen as representing the interests of these fiscal elite. Journalist David Graham Phillips wrote a series of articles decrying corruption and subservience to corporate interests within the body entitled Treason of the Senate.

La Follette believed his fears about the American economy were confirmed during the Bankers' Panic of 1907. La Follette opposed Aldrich's proposal (which had been created with the aid of financiers such as Paul Warburg). He saw the plan to issue $500 million in emergency currency backed in part by railroad bonds as an effort to establish economic centralization and crush free institutions. La Follette's troubles in the Senate worsened when fellow Progressive Theodore Roosevelt did not seek another term and william Howard Taft became President in 1909.

In 1909 La Follette founded La Follette's Weekly (later La Follette's Magazine) — a publication that continues in the 21st century as The Progressive.

In 1911, La Follette declared his intention to run for President and campaigned to mobilize the progressive elements in the Republican Party behind his bid. Mentally and physically exhausted, anxious over an impending operation on his thirteen-year-old daughter for tuberculosis, La Follette made a rambling speech in February 1912 before a gathering of leading magazine editors that caused many to doubt his sanity. Many of his supporters deserted him for Theodore Roosevelt, who had decided to run for President again. At the highly charged Republican Convention, La Follette received only 41 delegates' votes to eventual nominee william Howard Taft's 561.

He came in third behind incumbent Calvin Coolidge and Democratic candidate John W. Davis. La Follette won 17% of the popular vote, carried Wisconsin (winning its 13 electoral votes) and polled second in 11 Western states. His base consisted of German-Americans, railroad workers, the AFL labor unions, the Non-Partisan League, the Socialist Party, Western farmers, and many of the "Bull Moose" Progressives who had supported Roosevelt in 1912. La Follette's 17% showing represents the third highest showing for a third party since the American Civil War (only surpassed by Roosevelt's 27% in 1912 and Ross Perot's 19% in 1992, although unlike Roosevelt and La Follette, Perot did not carry any states), while his winning of his home state represents the most recent occasion any third party presidential candidate has carried a non-Southern state.

Perhaps the most controversial of Senator La Follette's positions were his opposition to American entry into World War I and, later, his critique of the wartime policies of President Woodrow Wilson. "More than any of the other objectors to war," writes Historian Thomas Ryley, "he remained a symbol of opposition to the conflict and to Wilsonian policies for prosecuting it." La Follette had cautiously supported most of Wilson's domestic program, but by 1916 he was becoming increasingly critical of the president's foreign policy.

His address was scheduled for October 6, 1917. His opponents in Congress manipulated the schedule so they would follow him and prevent rebuttal. The public, sensing drama, packed the viewing galleries, and the majority of Senators made sure they were present to hear all the speeches. Upon taking the floor, La Follette read in an unemotional detached manner a speech he had prepared defending free speech in wartime. Upon his conclusion there was a spontaneous outburst of applause that had to be gavelled into order. This speech is hailed as "a classic argument for free speech during time of war".

During the 1924 convention, La Follette was filmed by Lee DeForest in DeForest's Phonofilm sound-on-film process, along with Franklin D. Roosevelt, John W. Davis, and Al Smith. Following the 1924 election, the Progressive Party disbanded.

La Follette died in Washington, D.C., on June 18, 1925 of cardiovascular disease several months after the election. He was buried in the Forest Hill Cemetery on the near west side of Madison, Wisconsin.

After his death, his wife, Belle Case La Follette, remained an influential figure and Editor, watching their sons Philip and Robert enter politics. By the mid-1930s, the La Follettes had reformed the Progressive Party on the state level in the form of the Wisconsin Progressive Party. The party quickly, if briefly, became the dominant political power in the state, electing seven Progressive congressmen in 1934 and 1936. Their younger son, Philip La Follette, was elected Governor of Wisconsin; their older son, Robert M. La Follette Jr., succeeded his father as Senator, leading a caucus of Progressives composed of Progressive, Farmer-Labor, American Labor, and various Republican and Democratic Party congressional representatives.

La Follette Jr. returned to the Republican Party in 1946 to run as its nominee for the Senate, but he was defeated in the primary by Joe McCarthy. His grandson, Bronson La Follette, served as Wisconsin's attorney general in the 1960s.

La Follette has been called "arguably the most important and recognized leader of the opposition to the growing dominance of corporations over the Government" and is one of the key figures in Wisconsin's long history of political liberalism. In 1957, a Senate Committee selected La Follette as one of the five greatest U.S. Senators, along with Henry Clay, Daniel Webster, John C. Calhoun, and Robert A. Taft. A 1982 survey asking historians to rank the "ten greatest Senators in the nation's history" based on "accomplishments in office" and "long range impact on American history," placed La Follette first, tied with Henry Clay.

The Wisconsin Idea promoted the idea of grounding legislation on thorough research and expert involvement. To implement this program, La Follette began working with University of Wisconsin–Madison faculty. This made Wisconsin a "laboratory for democracy" and "the most important state for the development of progressive legislation". As governor, La Follette signed legislation that created the Wisconsin Legislative Reference Library (now Bureau) to ensure that a research agency would be available for the development of legislation.