Chondroitin sulfate (CS) is one of the main bioactive compounds in animal cartilage. In this study, the anti-inflammatory activity of sturgeon-derived chondroitin sulfate (SCS) was evaluated in the dextran sulfate sodium (DSS)-induced BALB/c mice model. Orally administration of SCS significantly alleviated the DSS-induced colitis symptoms, including the reduction of crypt depth, inhibition of the abnormal crypt foci formation, down-regulation of the proinflammatory biomarkers (NO, interleukin (IL)-6, IL-1β and tumor necrosis factor-α) and up-regulation of the anti-inflammatory biomarkers (IL-10 and IL-4). The gut microbiota analysis revealed that SCS alters the intestinal microbiota composition in colitis mice, especially the increase of the relative abundance of Ruminococcaceae and Lachnospiraceae. This alternation further induced primary bile acids convert into secondary bile acids. With SCS administration, the levels of deoxycholic acid (DCA) and litho cholic acid (LCA) were increased by 1.5- and 2.5-fold, respectively. The stimulated secretion of DCA and LCA showed further activation of the NF-κB signaling pathway, thereby suppressing the inflammatory response and attenuating inflammatory bowel disease (IBD) in mice. This study provided a valuable strategy for colitis prevention and treatment with sturgeon cartilage by-products.