Having played baseball throughout his youth, Spalding first played competitively with the Rockford Pioneers, a youth team, which he joined in 1865. After pitching his team to a 26-2 victory over a local men's amateur team (the Mercantiles), he was approached by another, the Forest Citys, for whom he played for two years. In the autumn of 1867 he accepted a $40 per week contract, nominally as a clerk, but really to play professionally for the Chicago Excelsiors, a not uncommon arrangement contrary to the rules of the time. Following the formation of the National Association (NA), baseball's first professional league, in , Spalding joined the Boston Red Stockings (precursor club to the modern Atlanta Braves) and was highly successful; winning 205 games (and losing only 53) as a pitcher and batting .323 as a hitter.
Spalding did not like the way the game was playing in the NA, so he decided to create another league. During the season, he secretly signed a contract that had him playing for the Chicago White Stockings (now known as the Chicago Cubs). At the same time he coaxed fellow players Deacon White, Ross Barnes and Cal McVey, as well as Cap Anson (while the first three were teammates of Spalding, Anson was from the Philadelphia Athletics of the NA). Just after this had all occurred, the plan became public. Due to Spalding signing with the White Stockings, he effectively ended the NA.
Meanwhile, he and his brother began a sporting goods store in Chicago. After the NA folded, he joined the Chicago White Stockings of the newly formed National League in 1876, winning 47 games as the club captured the league's inaugural pennant. In , Spalding began to use a glove to protect his hands. People had used gloves previously, but never had a star like Spalding.
Spalding published the first official rules guide for baseball. In it he stated that only Spalding balls could be used (previously, the quality of the balls used had been subpar.) Spalding also founded the Baseball Guide, which at the time was the most widely-read baseball publication. Spalding retired from playing baseball in 1878, although he continued as a major force as owner of the White Stockings and major influence on the National League. Spalding's .796 career winning percentage (from an era when teams played about once or twice a week) is the highest ever achieved by a baseball pitcher.
In 1888-1889, Spalding took a group of Major League players around the world to promote baseball and Spalding sporting goods. Playing across the western U.S., the tour made stops in Hawaii (although no game was played), New Zealand, Australia, Ceylon, Egypt, Italy, France, and England. The tour returned to grand receptions in New York, Philadelphia, and Chicago. The tour included future Hall of Famers Adrian "Cap" Anson and John Montgomery Ward. While the players were on the tour, the National League instituted new rules regarding player pay that led to a revolt of players, led by Ward, who started the Players' League the following season (1890). The league lasted one year, partially due to the competitive tactics of Spalding to limit its success.
Spalding's store grew rapidly over the next 25 years, with 14 stores by 1901, expanded from retail into manufacturing baseball equipment and is still a going concern. In 1900 Spalding was appointed by President McKinley as the USA's Commissioner at that year's Summer Olympic Games. In 1905, after Henry Chadwick wrote an article saying that baseball grew from the British sports of cricket and rounders, Spalding called for a commission to find out the real source of baseball. The commission called for citizens who knew anything about the founding of baseball to send in letters. After 3 years of searching, on December 30, 1939. His nephew, also named Albert Spalding, was a renowned violinist.