〔Abstract〕 ObjectiveTo explore the function and related molecular mechanisms of magnoflorine against colon cancer vianetwork pharmacology, molecular docking, and in vitro cell experiments. Methods In this study, the canonical SMILES of magnoflorine was obtained from the PubChem database, and the potential targets of magnoflorine were predicted by the Swiss Target Prediction database, while the disease targets of colon cancer were obtained from the DisGeNET, GeneCards and OMIM databases. The intersecting targets between magnoflorine’s predicted targets and colon cancer disease targets were taken, and a protein-protein interaction (PPI) network was constructed and analyzed. The DAVID online database was employed to conduct Gene Ontology (GO) and KEGG pathway enrichment analyses on core targets. The top five key targets screened were docked with magnoflorine using AutoDock software. Finally, cellular experiments including CCK-8 assays, EdU experiments, cell scratch assays, and Transwell assays were conducted to validate the results from network pharmacology and molecular docking. Results44 key targets of magnoflorine in resisting colon cancer were acquired. The molecular docking results showed that magnoflorine had a strong binding activity with the core target signal transducer and activator of transcription 3 (STAT3) in the top five of the PPI network. Cellular experiments confirmed that magnoflorine could inhibit the proliferation and migration of colon cancer cells by suppressing the JAK/STAT3 signaling pathway. ConclusionMagnoflorine may inhibit the proliferation and migration of colon cancer cells by regulating the JAK/STAT3 signaling pathway.