Objective To investigate the mechanism of action of effective active components of Agrimoniae Herba in the treatment of ulcerative colitis(UC) in vitro. Methods We screened the effective active ingredients of Agrimoniae Herba and obtained the Agrimoniae Herba-UC intersecting targets through the database, analyzed the KEGG and GO pathway enrichment of the targets, and screened the tightly bound target proteins through molecular docking. In vitro RAW264.7 and Caco-2 cell models were constructed to verify the effects of the screened active ingredients on inflammatory factors, macrophage polarization and intestinal tight junction protein expression. Results The main active ingredients of Agrimoniae Herba were lignans, kaempferol, quercetin, catechin, and ellagic acid, which significantly regulated macrophage polarization and the expression of inflammatory factors IL-1β, IL-6, TNF-α, iNOS, and CD86, but did not significantly improve intestinal tight junction proteins. The important target genes were MMP3, MMP2, BCL2L1, CD40, and FOS, which were mainly enriched in the signaling pathways such as NF-κB, PI3K-Akt, MAPK, HIF-1, TGF-β, and so on. Conclusion The active ingredients of Agrimoniae Herba can reduce the inflammation level and regulate macrophage polarization, and the mechanism may be through the regulation of MMP3, MMP2, BCL2L1, CD40, FOS, and other genes, thereby regulating the signaling pathways such as NF-κB, PI3K-Akt, MAPK, HIF-1, TGF-β, and so on, to achieve the effect of treatment of UC.