Little was born in New Haven, Connecticut on July 4, 1942. He attended the Hillhouse High School in New Haven and the Bordentown Military Institute in Bordentown, New Jersey.

Little was recruited by Gen. Douglas MacArthur to play football at the United States Military Academy and had told him that he’d ascend to the rank of general if he enrolled at West Point. He was also recruited by Notre Dame. Little ultimately chose to attend Syracuse at the persuasion of first African-American Heisman winner Ernie Davis. Little is the only three-time All-American running back to compete for the Syracuse University Orangemen. He finished 5th in Heisman Trophy voting in both 1965 and 1966.

In 1975, Little retired as the NFL’s 7th all-time leading rusher with 6,323 yards rushing and 54 total touchdowns (rushing, receiving and returns). He also threw a touchdown pass to receiver Jerry Simmons in a 1972 upset over the Oakland Raiders. During his rookie year, Little led the NFL in punt returns with a 16.9-yard average. He led the NFL in combined yards in 1967 and 1968. Little was Denver Broncos team captain all 9 seasons, including his rookie season.

Little lives with his wife DeBorah in Las Vegas. Little finished 40th in his class of 140 at the University of Denver law school, from which he received his masters in legal administration degree in 1975. Little owned automobile dealerships in Denver, the Seattle area and Santa Barbara. In May 2020, former Syracuse teammate Pat Killorin announced that Little had been fighting cancer.

Little was a charter member of the Broncos Ring of Fame in 1984, which included Rich Jackson, Lionel Taylor and Goose Gonsoulin. He was the first Bronco to win a rushing title, leading the AFC in rushing in 1970 with 901 yards and the following year he became the first Bronco to eclipse 1,000 yards, gaining 1,133 to lead the NFL. Little was the first player to lead his conference in rushing for a last place team and the 13th player ever in professional football to rush for at least 1,000 yards in one season. He was an American Football League All-Star in 1968. In a week 12 win over Buffalo, he caught 4 passes out of the backfield for 165 yards, including a 66-yard touchdown, setting a franchise record of 41.25 yards per reception that still stands. He was named first-team “All-AFL” in 1969, and made the AFC-NFC Pro Bowl in 1970, 1971 and 1973. At 5′10″ and 195 pounds, Little was the smallest back to lead the league in rushing since World War II. He led the league in combined yards in 1967 and 1968 and was the only player to return punts for touchdowns in both seasons. During a 6-year period, 1968–1973, Little rushed for more yards and more yards from scrimmage (rushing and receiving) than any RB in the NFL.

Floyd Little is portrayed by the late actor Chadwick Boseman in the 2008 Universal Pictures film The Express: The Ernie Davis Story, a biographical film about Syracuse University Heisman Trophy winner Ernie Davis.

In 2009 Little was a finalist for induction into the Hall of Fame. He was voted in on February 6, 2010, his induction took place in Canton, OH on August 7, 2010.

From July 2011 to June 2016, Little served as the Special Assistant to the Athletic Director at Syracuse University.