Chondroitin sulfate (CS) has been widely used for medical and nutraceutical purposes due to its roles in maintaining tissue structural integrity. We investigated if CS disaccharides may act as a bioactive compound and modulate gut microbial composition in mice. Our data show that CS disaccharides supplementation for 16 days significantly reduced blood LPS in the mice experiencing exhaustive exercise stress. CS disaccharides partially restored total fecal short-chain fatty acids from the level significantly repressed in mice under the stress. Our findings demonstrated that CS was likely butyrogenic and resulted in a significant increase in fecal butyrate concentration. CS disaccharides had a profound impact on gut microbial composition, affecting the abundance of 13.6% and 7.3% Operational Taxonomic Units in fecal microbial communities in healthy and stressed mice, respectively. CS disaccharides reduced the prevalence of inflammatory Proteobacteria. Together, our findings demonstrated that CS may ameliorate stress-induced intestinal inflammation. Furthermore, CS significantly increased intestinal Bacteroides acidifaciens population, indirectly exerting its immunomodulatory effect on the intestine. CS disaccharides had a significant impact on a broad range of biological pathways under stressed condition, such as ABC transporters, two-component systems, and carbohydrate metabolism. Our results will facilitate the development of CS as a bioactive nutraceutical.