This study investigates the therapeutic mechanisms of cambogin (Cam) in treating ulcerative colitis (UC) using network pharmacology, molecular docking, and in vitro experiments. Initially, the intersection targets of Cam and UC were predicted and screened using databases. Subsequently, GO analysis and KEGG analysis were employed to enrich core targets and pathways. Molecular docking was then performed, and an in vitro model of UC was established by inducing NCM460 human normal colonic epithelial cells with 10 μg/mL lipopolysaccharide to verify the anti-inflammatory effects of Cam. The results of network pharmacology revealed that there were 42 potential targets for Cam in treating UC. GO enrichment analysis indicated that Cam was involved in biological processes such as inflammatory response and protein phosphorylation. KEGG pathway enrichment analysis demonstrated that Cam exerted its anti-ulcerative colitis effects by participating in the FOXO signaling pathway. The results showed that Cam significantly reduced the levels of nitric oxide (NO), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and reactive oxygen species (ROS) in the UC model (P<0.05 or P<0.01). Molecular docking results indicated that Cam had good binding affinity with core targets in the FOXO signaling pathway, including phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and forkhead box protein O4 (FoxO4). Western blot and RT-qPCR analyses confirmed that Cam decreased the phosphorylation levels of PI3K and AKT and mRNA expression of PI3K and AKT, while increasing the protein expression of FoxO4 and mRNA levels of FoxO4 (P<0.05 or P<0.01). It has been suggested that the therapeutic mechanism of Cam in the treatment of ulcerative colitis may be associated with its ability to inhibit the secretion of inflammatory cytokines, alleviate oxidative stress, and regulate the PI3K/AKT/FoxO4 signaling pathway.