Having played baseball throughout his youth, Spalding first played competitively with the Rockford Pioneers, a youth team, whom 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, baseball's first professional league, in 1871, Spalding joined the Boston Red Stockings (not a percursor club to the modern Red Sox) and was highly successful; winning 205 games (and losing only 53) as a pitcher and batting .323 as a hitter. 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. Spalding retired from baseball two years later.

Retired from the game, he and his brother opened a sporting goods store in Chicago, obtaining the rights to produce the official National League ball. The business, which 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. Seven years later, his prompting would lead to the founding of the commission that (erroneously) declared baseball to be the invention of Abner Doubleday.

Receiving the archives of the late Henry Chadwick in 1908, Spalding combined these records with his own memories (and biases) to write Americas National Game (published 1911) which, despite its flaws, was probably the first scholarly account of the history of baseball.

He was elected to the Baseball Hall of Fame by the Old Timer's Committee in 1939.