To identify the key targets of atractylenolide Ⅲ(AT-Ⅲ) and to explore the mechanism of actin-related protein 2/3(Arp2/3) in inhibiting ulcerative colitis(UC).Methods:Characteristic proteins of rat small intestinal crypt epithelial cells(IEC-6) were screened using limited proteolysis-mass spectrometry(lip-SMap).Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were conducted to determine the key targets of AT-Ⅲ.An UC model was simulated by co-incubating lipopolysaccharide(LPS) with IEC-6 cells.Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of interleukin-6(IL-6) and tumor necrosis factor-alpha(TNF-α).A scratch assay was performed to measure the migration rate of IEC-6 cells.Western blot was used to measure the levels of E-cadherin，vimentin，and Arp2/3.Molecular docking was conducted to evaluate the binding ability of AT-Ⅲ to its key targets.Results:The characteristic proteins obtained from lip-SMap were involved in regulating the phosphatidylinositol 3-kinase-serine/threonine kinase(PI3K-AKT) signaling pathway and the tight junction(TJ) signaling pathway.Compared with the control group，the LPS group showed increased levels of IL-6 and TNF-α(both P<0.05)，an increased migration rate(P<0.05)，and various impacts on the expression of E-cadherin and vimentin(both P<0.05).Compared with the LPS group，the LPS+CK666+AT-Ⅲ group significantly reduced IL-6 and TNF-α levels(both P<0.01)，effectively reversed LPS-induced changes in cell migration and the expression of E-cadherin and vimentin(both P<0.05).Molecular docking showed that Arp2/3 had good binding activity with AT-Ⅲ.Conclusion:Arp2/3 is a key target of AT-Ⅲ and can alleviate LPS-induced UC through epithelial-mesenchymal transition(EMT).