Derivation of Qingchang Huashi formula, named Qingchang Huashi Jianpi Bushen (QCHS_JPBS) formula, has shown significant therapeutic effect on patients with ulcerative colitis (UC). In this study, the potential mechanism of QCHS_JPBS formula in repairing mucosal damage was explored from the perspective of intestinal stem cell (ISCs) differentiation, and potential targets of the QCHS_JPBS formula to improve UC were predicted using network pharmacology analysis. The therapeutic efficacy of QCHS_JPBS formula was evaluated in a mouse model of 2.5% dextran sulfate sodium (DSS) induced colitis. The effect of this formula on the ISC differentiation was evaluated using tissue transmission electron microscopy, immunofluorescence, and RT-qPCR. The cecal contents were subjected to 16s RNA sequencing analysis and non-target metabolomics analysis using LC-MS/MS. The fecal microbiota transplantation method verified the essential role of gut microbiota in promoting ISC differentiation and repairing mucosal damage. The results indicated that QCHS_JPBS formula suppressed the inflammatory response and repaired the damaged intestinal epithelial barrier in DSS-induced colitis mice. QCHS_JPBS formula promoted ISC differentiation, particularly in the direction of goblet cells. QCHS_JPBS formula restored gut dysbiosis and regulated metabolic disorders in DSS-induced colitis mice. And then, the results of fecal microbiota transplantation indicated that QCHS_JPBS formula promoted differentiation of intestinal stem cells to repair mucosal damage through gut microbiota. Finally, a total of 79 active ingredients of QCHS_JPBS formula were identified based on LC-MS analysis and EGFR, STAT3, SRC, AKT1, and HSP90AA1 were considered as potential therapeutic UC targets of QCHS_JPBS formula based on network pharmacology analysis. The present study demonstrated that QCHS_JPBS formula promoted the differentiation of ISCs through gut microbiota to repair the damaged intestinal epithelial barrier in UC mice.