This study investigated the protective effects of Garcinia biflavonoid 1 (GB1) against dextran sulfate sodium (DSS)-induced ulcerative colitis and its underlying mechanisms. Using wild-type (WT) and NLRP3 knockout (Nlrp3-/-) mice, we demonstrated that GB1 administration significantly ameliorated colitis symptoms, as evidenced by improved body weight, disease activity index (DAI) scores, colon length, and histological damage in WT mice. Mechanistically, GB1 downregulated pro-inflammatory mediators (IL-6, NF-κB, and CD11b) while attenuating the expression of NLRP3 inflammasome components (ASC, Caspase-1, and IL-1β). Notably, these protective effects were abolished in Nlrp3-/- mice, confirming the essential role of NLRP3 in GB1-mediated mitigation of colitis. Furthermore, GB1 reinforced intestinal barrier integrity by preserving tight junctions, reducing permeability, and attenuating mucosal inflammation. Collectively, our findings highlight GB1 as a promising therapeutic candidate for colitis treatment, primarily through NLRP3 inflammasome suppression and intestinal barrier restoration.