ObjectiveThe objective of this study was to assess the pharmacological activity and therapeutic mechanism of Dahuang Mudan Decotion (DHMDD) for colorectal cancer using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS), network pharmacology and in vitro experiments.MethodsThe chemical components of DHMDD were identified by UPLC-MS. Network pharmacological analysis was utilized to screen the active ingredients and targets associated with DHMDD for colorectal cancer. Based on the results of network pharmacology, the potential mechanism of DHMDD on colorectal cancer predicted was experimentally studied and verified in vitro.ResultsDHMDD primarily exerts its effects on colorectal cancer through 52 active ingredients. AKT1, ESR1, HSP90AA1, JUN, PIK3CA, PIK3CB, PIK3R1, SRC, STAT3, TP53 were the top 10 targets. The top 10 ingredient nodes were Quercetin, Physcione, Pontigenin, Crysophanol, Linolenic acid, Piceatannol, Adenosine, Emodin, Sambunigrin, and Prunasin. The main compounds and the target proteins exhibited strong binding ability in molecular docking studies. The results of cell experiments demonstrated that DHMDD can inhibit the proliferation, invasion and migration of CRC cells through the PI3K/Akt pathway.ConclusionThrough network pharmacology analysis and cell experiments, this study suggests that DHMDD can exert its therapeutic effects on colorectal cancer through a combination of multiple components and targets.