Colorectal cancer (CRC) is the most prevalent cancer of the digestive tract. Herba Patriniae (also known as Bai Jiang Cao, HP) have been widely used to manage diarrhea, ulcerative colitis, and several cancers, including CRC. Nonetheless, the molecular mechanisms underlying the pharmacological action of HP on CRC remain unclear. This study investigated the underlying mechanisms of HP against CRC using network pharmacology analysis and in vitro and in vivo experiments. The results revealed nine bioactive compounds of HP. Furthermore, 3460 CRC-related targets of the identified active compounds were predicted from the Gene Expression Omnibus (GEO) database. Furthermore, 65 common targets were identified through the intersection of two related targets. Moreover, ten hub genes, including CDK4, CDK2, CDK1, CCND1, CCNB1, CCNA2, MYC, E2F1, CHEK1, and CDKN1A were identified through the topological analysis. Meanwhile, the GO and KEGG pathway analysis revealed that the core target genes were majorly enriched in the p53 and HIF-1 signaling pathways. Moreover, HP promoted apoptosis and suppressed cell proliferation by activating the p53 signaling pathway in a dose-dependent manner, while a similar effect was observed for Isovitexin (the primary component of HP). Overall, this study provides valuable insights into the underlying mechanisms of HP and its component Isovitexin against CRC, providing a theoretical foundation for additional experimental verification of its clinical application.