Background: Ulcerative colitis (UC) is the chronic inflammatory bowel disease with the feature of gut microbial dysbiosis and dysregulation of intestinal homeostasis. Costunolide (COS), an important active component of the traditional Chinese medicine Radix Aucklandiae, is shown with therapeutic effects on UC. However, it is unclear whether COS improves UC by regulating imbalances in gut microbiota and facilitation of repair in the intestinal mucosa. Hererin, the present work focused on investigating function of COS in regulating gut microbiota and mucosal repair, for further understand the mechanism underlying Radix Aucklandiae’s therapeutic effects on UC. Results: Network pharmacology analysis revealed that COS acts on different targets to intervene in inflammation, immunity, apoptosis, and other pathways to treat UC. Through the utilization of microbiota analysis according to 16S rRNA gene, we initially observed a significant beneficial effect of COS on the dysregulated gut microbiota in colitis-afflicted mice. The treatment of COS resulted in an augmentation of bacterial diversity, decreased Proteobacteria, Bacteroidetes, and Verrucomicrobiota proportions, and increased Firmicutes and Actinobacteria abundances. At the level of genus, COS reduced Helicobacter abundance and enhanced Bacteroides, Lactobacillus, and Allobaculum abundances. Furthermore, COS significantly improved intestinal mucosal damage repair processes by increasing the gene expression of intestinal repair factors regulators such as klf5, Elmo1, fprl, CD274, and Lyz2, and through elevating goblet cell quantity responsible for mucus production. Additionally, COS modulated colonic tight junction (Tj) protein levels, as evidenced by increased protein levels of claudin and Occludin. These findings represent the first documented evidence of COS’ efficacy in promoting intestinal mucosal repair. Conclusions: Our findings suggest that COS may improve UC by regulating gut microbiota dysbiosis and promoting intestinal mucosal repair. This study provides further insights into the mechanism underlying Radix Aucklandiae’s therapeutic effects on UC, and highlights the potential of COS as the promising anti-UC treatment.