Ulcerative colitis (UC), a chronic inflammatory bowel disease (IBD), presents therapeutic challenges due to the limitations and side effects of current treatments, creating a need for safer and more effective alternatives. Despite several studies on the health benefits of donkey milk, its specific impact on UC remains unsolved. This study investigates the therapeutic potential of donkey whey protein (DWP), a bioactive-rich functional food, in alleviating UC symptoms in a mouse model of dextran sodium sulfate-induced colitis. The DWP was processed using thermal and nonthermal techniques, including high-temperature long-time (HTLT) pasteurization, ultrasonic processing (300 W), and UV irradiation, to evaluate the impact of processing methods on its therapeutic efficacy. The results demonstrated that when processed nonthermally, DWP mitigated colitis symptoms such as weight loss, diarrhea, and colon shortening. Histological analysis revealed improved mucosal integrity and reduced inflammation in the colon, with nonthermal treatments showing superior effects. The DWP treatment enhanced antioxidant activity, suppressed proinflammatory cytokines (TNF-alpha, IL-6, IL-1beta), and modulated the gut microbiota, promoting microbial diversity and restoring microbial balance. Nonthermal treatments were particularly effective in preserving the bioactivity of DWP, highlighting their potential in enhancing the functional properties of whey proteins. These findings suggest that DWP, particularly when processed through nonthermal methods, holds potential as a natural and effective alternative for managing UC and other chronic inflammatory diseases.