BACKGROUND: The progression of inflammatory bowel disease (IBD) is closely connected with intestinal flora dysbiosis. Sakuranetin (SAK) is a natural compound with anti-inflammatory and antibiosis activities. We investigated the properties and mechanisms of SAK on IBD-like colitis. METHODS: Mice with dextran sulfate sodium (DSS)-induced colitis were accomplished to assess the effects of SAK on colitis, as well as intestinal mucosal immune imbalance and intestinal barrier dysfunction. 16S rDNA was used to characterize the intestinal flora, and the short-chain fatty acid (SCFA) content in faeces was calculated using GS‒MS. Faecal microbiota transplantation (FMT) and a pseudosterile model (antibiotic cocktail, ABX) were used to evaluate whether the effects of SAK on colitis were dependent on the gut flora. Pathohistological and biochemical tests were used to estimate the safety of SAK. RESULTS: SAK significantly ameliorated DSS-induced colitis in mice, verified by decreased weight loss, less colon shortening, and lower disease activity, histology and colonoscopy scores. Moreover, SAK alleviated gut dysbiosis and elevated the abundance of SCFA-producing bacteria in DSS-treated mice. Meanwhile, SAK increased faecal SCFA levels and activated GPR41/43 signalling. SAK also improved Treg/Th17 homeostasis and intestinal barrier function. In addition, ABX and FMT experiments confirmed that the ability of SAK to alleviate colitis was mediated through the gut flora. Finally, a safety experiment revealed that SAK had no significant adverse effects on major organ or liver/kidney function. CONCLUSIONS: SAK may improve the intestinal immune balance and barrier function by regulating intestinal flora dysbiosis and increasing SCFA production, thereby protecting against colitis.