Taraxasterol (TAX) has been proven to prevent and treat inflammatory diseases. However, the effects of TAX on intestinal barrier and the diversity, structure, and function of gut microbiota have yet to be elucidated in dextran sodium sulfate (DSS)-induced colitis mice. Our objectives are to evaluate the effect of TAX on intestinal barrier and its impact on gut microbiota. Herein, immunofluorescence analysis is conducted to determine the expressions of tight junction (ZO-1) and mucin (Mucin-2) proteins. The abundance, diversity, and function of fecal colonies are investigated by using 16S rDNA sequencing, and the influence of TAX on the gut microbiota in mice is also analyzed. Our results suggest that TAX attenuates the symptoms in DSS-induced colitis mice by reducing the DAI score, increasing colon length, alleviating histopathological damage of colon tissues, and improving intestinal barrier. 16S rDNA sequencing of fecal samples indicates that TAX intervention has a regulatory effect on DSS-induced gut microbiota dysbiosis at different taxonomic levels. TAX increases microbial diversity that is reduced by DSS. It normalizes the relative abundance of and the ratio of /. In addition, treatment with TAX has a better effect on the function of metabolisms, such as nucleotide, lipid, and bile acid metabolism. These findings suggest that TAX may be a good candidate for the remission of colitis, which is related to improving intestinal barrier and modulating gut microbiota.