The pursuit of food-based alternatives to conventional therapies for ulcerative colitis (UC) demands immediate attention. In prior investigations, we synthesized WPI-stachyose conjugates through the Maillard reaction, identifying them as functional prebiotics. However, their impact on in vivo regulation of gut microbiota remains inadequately explored. To bridge this gap, we delved into the therapeutic effects and mechanisms of WPI-stachyose conjugates as prebiotic-functional components in C57BL/6J mice afflicted with dextran sodium sulfate (DSS)-induced UC. The treatment involving WPI-stachyose conjugates led to significant therapeutic advancements, evident in the reduction of pro-inflammatory cytokine levels and restoration of gut microbiota composition. Noticeable enhancements were observed in UC-associated symptoms, including weight loss, colon length reduction, and tissue damage, notably improving in the treated mice. Remarkably, both the conjugates and the physical combination effectively lowered pro-inflammatory cytokines and oxidative stress, with the conjugates demonstrating enhanced effectiveness. Furthermore, the simultaneous administration of WPI-stachyose conjugates further amplified the presence of beneficial bacteria and elevated short-chain fatty acids, acknowledged for their favorable impact across various conditions. These findings underscore the potential therapeutic application of WPI-stachyose conjugates in addressing DSS-induced UC, offering insights into innovative therapeutic strategies.