Mulberry has attracted wide attention due to its substantial nutritional values. This work first studied the protective effect of mulberry anthocyanins (MAS) on dextran sulfate sodium (DSS)-induced colitis. The mice experiment was designed as four groups including normal mice (Control), dextran sodium sulfate (DSS)-fed mice, and DSS plus 100 mg/kg·bw MAS-fed mice (LMAS-DSS) or DSS plus 200 mg/kg·bw MAS-fed mice (HMAS-DSS). Mice were given MAS by gavage for 1 week, and then DSS was added to the drinking water for 7 days. MAS was administered for a total of 17 days. The results showed that oral gavage of MAS reduced the disease activity index (DAI), prevented colon shortening, attenuated colon tissue damage and inflammatory response, suppressed colonic oxidative stress and restored the protein expression of intestinal tight junction (TJ) protein (ZO-1, occludin and claudin-3) in mice with DSS-induced colitis. In addition, analysis of 16S rRNA amplicon sequences showed that MAS reduced the DSS-induced intestinal microbiota dysbiosis, including a reduction in Escherichia-Shigella, an increase in Akkermansia, Muribaculaceae and Allobaculum. Collectively, MAS alleviates DSS-induced colitis by maintaining the intestinal barrier, modulating inflammatory cytokines, and improving the microbial community.