The Witte Museum opened in 1926 under the charter of the San Antonio Museum Association, a nonprofit corporation established in 1925. Ellen Schulz, a local school teacher, had raised $5,000 to purchase an extensive private collection of natural history specimens for the city of San Antonio. The collection was first housed in the local high school, and when it outgrew this space, Miss Schulz and her supporters petitioned city commissioners to build a new museum. The building was constructed with public funds and a $65,000 bequest to the city from local businessman Alfred G. Witte, who stipulated that a museum be built in Brackenridge Park in memory of his parents. The facility was known as the Witte Memorial Museum until 1984, when the name was simplified to the Witte Museum.
The museum survived financial struggles during the Great Depression and World War II through activities that included operation of a reptile garden, a popular natural history attraction adjacent to the museum. In the 1930s the Witte conducted a series of archeological excavations in the lower Pecos region of Texas, forming the basis for one of its most important collections. The museum also served as a teaching institution, offering art and natural history classes for all ages. Continued growth of the museum's permanent collection and exhibits necessitated building additions in 1936, 1949, 1958, and 1962. Three historic houses, the Twohig House, the Ruiz House, and the Navarro House, were relocated to the Witte's grounds in the 1940s, and two reproduction log buildings were constructed.
From 1969 until 1987 the museum association operated the Witte Confluence Museum (later called the San Antonio Museum of Transportation), to exhibit its horse-drawn carriage and antique car collection. The museum was located in HemisFair Plaza and closed when the oil and gas recession adversely impacted the association's funding. The Witte Museum's main emphasis shifted to art in 1970 when an art historian was hired as director. Subsequently, the museum association purchased and renovated an abandoned nineteenth-century brewery to house its third museum, the San Antonio Museum of Art. When the Museum of Art opened in 1981, the Witte refocused on its original purpose, to exhibit natural history, anthropology, and Texas and regional history. As the audience, programs, and exhibits of the Witte Museum and San Antonio Museum of Art increased and diversified in the 1980s, more specialized staff was required to meet the programming needs of these two institutions. The Witte Museum was accredited by the American Association of Museums in 1974 and reaccredited in 1991.
In 1992 the San Antonio Museum Association's board of trustees initiated reorganization to establish two independent museums, and this process was completed in May 1994. Major improvements and expansions of the Witte campus continued during the 1990s, and in 1997 the museum opened the H-E-B Science Treehouse, designed to provide hands-on science activities for children and adults. By 2003 a major upgrade of the Witte gallery’s climate control system was completed.
On May 26, 2012, the Witte Museum opened the Robert J. and Helen C. Kleberg South Texas Heritage Center. This 20,000-square-foot, two-story building that incorporates the historic Pioneer Hall (located next to the original Witte Museum), permanently houses the Witte’s South Texas collections, exhibitions, and public programs. The South Texas collections include saddles, spurs, basketry, branding irons, historical clothing, firearms, land grants, and art and chronicles the stories of Tejano freighters, chili queens, Texas Indians, Spanish settlers, farmers and ranchers, oilmen, cowboys, women, children, and others that comprise the heritage of South Texas. The center includes a Texas art gallery, outdoor amphitheater, sculptures, and other features.
The Witte undertook the transformation of the H-E-B Science Treehouse into the H-E-B Body Adventure, promoted as the “first interactive health experience in the United States”; it opened in 2014. That same year the B. Naylor Morton Research and Collections Center, which provided exhibit space for more than 300,000 artifacts as well as space for scholarly, archival, and school activities, opened. The Mays Family Center for special events and exhibitions opened in 2016. Major renovations continued, and on March 4, 2017, the “New Witte”—the result of a $100 million effort encompassing 174,000 square feet—opened. Features include a new main building façade and entryway, the Naylor Family Dinosaur Gallery, Kittie West Nelson Ferguson People of the Pecos Gallery, as well as the Zachry Family Acequia Garden outside near the San Antonio River. Popular exhibits in 2018 included the Witte’s celebration of the Tricentennial of San Antonio with Confluence and Culture: 300 Years of San Antonio History, which opened in March and Predators vs Prey: Dinosaurs on the Land Before Texas, which opened in May.
The Witte Museum is the legal successor to the San Antonio Museum Association. The Witte Museum's collections, programs, and exhibits focus on the areas of history, science, and the humanities. The permanent collection represents ethnography, decorative arts and textiles, science, natural sciences with a particular emphasis on South Texas, and the history of Texas and the Southwest. Educational programs include workshops, demonstrations and live gallery theater performances for young people and families, spring break and summer camps for children, in-depth Humanities Center presentations for adult audiences, and Elderhostel programs for older adults. Ellen D. Schulz Quillin was the Witte Museum's director from 1926 until 1960. She was succeeded by William A. Burns in the 1960s, Jack R. McGregor in the 1970s, Mark Lane (1982–95), Linda K. Johnson (1995–98), and James C. McNutt (1999–2003). In 2004 Marise McDermott was appointed president and CEO of the Witte and continued in that capacity in 2020.