Tetrastigma hemsleyanum, a precious edible and medicinal plant in China, has attracted extensive research attention in recent years due to its high traditional value for the treatment of various diseases. In vitro digestion and colonic fermentation models were established to evaluate the stability of Tetrastigma hemsleyanum leaves (THL) phenolics by the HPLC-QqQ-MS/MS method. The total phenolic and flavonoid contents were degraded during digestion and fermentation. 3-caffeoylquinic acid, 5-caffeoylquinic acid, orientin and (iso)vitexin were metabolized by digestive enzymes and the gut microbiota, and absorbed in the form of glycosides and smaller phenolic acids for hepatic metabolism. The protective effects of THL on dextran sodium sulfate (DSS)-induced colitis in mice and potential mechanisms were explored. The results showed that THL supplementation increased the body weight and colon length, and the expression levels of tight junction proteins including occludin, claudin-1 and ZO-1 were up-regulated by THL. The secretions of pro-inflammatory cytokines containing IL-1β, IL-6 and TNF-α were significantly suppressed, whereas the content of anti-inflammatory cytokine IL-10 was promoted in the THL treated group. In addition, THL treatment activated the nuclear transfer of Nrf2, improved the expression of SOD, CAT, HO-1, NQO1 and GCLC, and decreased the content of MPO and MDA. It is worth noting that THL treatment significantly increased the content of short-chain fatty acids (SCFAs), increased the abundance of Ruminococcaceae, and decreased the abundance of Verrucomicrobia which is positively correlated with pro-inflammatory cytokines. These results indicated that THL effectively inhibited DSS-induced colitis by maintaining the intestinal epithelial barrier, mitigated oxidative stress through regulating the Keap1/Nrf2 signaling pathway and regulated the imbalance of the intestinal flora structure.