Ulcerative colitis (UC), a common inflammatory bowel disease, has become increasingly prevalent worldwide, posing significant health challenges. This study explored the anti-inflammatory effects of beta-sitosterol on UC and its underlying molecular mechanisms. Using a dextran sulfate sodium (DSS)-induced colitis model in male C57BL/6 mice, the therapeutic potential of beta-sitosterol at low (2 mg/kg) and high (6 mg/kg) doses was compared with sulfasalazine (300 mg/kg) as a positive control. Disease progression was assessed through Disease Activity Index (DAI) scores, histological analysis, and inflammatory marker expression. beta-sitosterol significantly ameliorated colonic inflammation, demonstrated by lower DAI scores, improved histological architecture, and reduced levels of inflammatory mediators, including NO, MPO, IL-6, and iNOS, while upregulating the anti-inflammatory cytokine IL-10. Mechanistically, beta-sitosterol promoted AMP-activated protein kinase (AMPK) expression and suppressed myosin light chain kinase (MLCK) expression. These findings were validated in vitro using LPS-stimulated Caco-2 cells, where beta-sitosterol decreased inflammatory marker levels and modulated AMPK/MLCK signaling. Notably, the use of Compound C, an AMPK inhibitor, reversed these effects by suppressing AMPK activity and restoring MLCK expression, confirming that the anti-inflammatory actions of beta-sitosterol are AMPK-dependent. In conclusion, this study highlights the therapeutic potential of beta-sitosterol in UC through modulation of the AMPK/MLCK signaling pathway. These findings not only deepen our understanding of beta-sitosterol's anti-inflammatory properties but also suggest its potential in developing novel AMPK-targeted therapies for inflammatory bowel disease management.