Magnoflorine (Mag), a natural alkaloid component originating from the Ranunculaceae Juss. Family, has a various of pharmacological activities. This study aimed to investigate the therapeutic effects and potential mechanism of Mag on dextran sulfate sodium (DSS)‐induced ulcerative colitis (UC) based on comprehensive approaches. Therapeutic effects of Mag on 3% DSS‐induced UC mice were analyzed. UHPLC‐Q‐TOF/MS was performed to investigate the potential metabolites and signaling pathway of Mag on DSS‐induced UC. Furthermore, the predicted mRNA and protein levels of JAK2/STAT3 signaling pathway in colon tissue were verified and assessed by qRT‐PCR and Western Blotting, respectively. Therapeutic effects of Mag on UC mice were presented in down‐regulation serum biochemical indices, alleviating histological damage of colon tissue. Serum untargeted metabolomics analysis showed that the potential mechanism of Mag on UC is mainly associated with the regulation of six biomarkers and 11 pathways, which may be responsible for the therapeutic efficacy of UC. The “component–metabolites–targets” interactive network indicated that Mag exerts its anti‐UC effect by regulating PTGS1 and PTGS2, thereby regulating arachidonic acid. Moreover, the results of qRT‐PCR showed that Mag could substantially decrease the relative mRNA expression level of Hub genes. In addition, it was found that Mag could inhibit the relative mRNA and protein expression of JAK2/STAT3 signaling pathway. The present results highlighted the role of Mag ameliorated colon injury in DSS‐induced UC mice by inhibiting the JAK2/STAT3 signaling pathway. These results suggest that Mag may be an effective agent for the treatment of UC.