Objective To observe the regulatory effects of sanguinarine (Sang) on proliferation and migration of bladder cancer T24 cells, and to explore the possible target genes and mechanisms of action. Methods Bladder cancer T24 cells and human normal bladder epithelial cells SV-HUC-1 were cultured and divided into the experimental and control groups, respectively. Cells in the experimental group were added with 0. 012 5, 0. 025, 0. 05, 0. 1, 0. 2, 0. 4, 0. 8, and 1. 6 μmol/L of Sang, and no Sang was added in the medium of the control group, and the proliferation ability of the cells was detected by CCK-8 kit. The half inhibitory concentration (IC50) was calculated. T24 cells were divided into experiment and control groups; cells in the experimental group were given Sang, cells in the control group were not treated with Sang, and the cell migration ability was detected by Transwell assay. Based on BATMAN, SwissTargetPrediction network pharmacology database and TCGA database, we obtained the intersection target genes of Sang and bladder cancer therapeutic targets, respectively, performed GO analysis and KEGG functional enrichment analysis, mapped the protein interaction network, and screened key genes of Sang in the regulation of T24 cells. GSEA_4. 2. 3 was used for key gene set enrichment analysis to identify the relevant pathways for key gene set enrichment. The differences in progression-free interval, disease-specific survival, and overall survival between the high and low expression groups of key genes were evaluated based on the GEPIA website and the "survival" R software package. Molecular docking of the Sang and key gene protein structures was performed and the binding energy was calculated. Real-time fluorescence quantitative PCR was used to detect key target genes in cells of experimental and control groups. Results The proliferative capacity and the number of membrane-penetrating cells in T24 cells of the experimental group were lower than those in the control group (both P <0. 05). The IC50 of Sang was 0. 200 3 μmol/L for T24 cells and 0. 709 9 μmol/L for SV-HUC-1 cells. The key genes regulated by Sang in T24 cells were screened as matrix metalloproteinase (MMP)-2, MMP-9, and prostaglandin-endoperoxide synthase 2 ( PTGS2). MMP-2, MMP-9, and PTGS2 were significantly associated with the pathway regulating the proliferation of epithelial cells. The disease-specific survival and overall survival of those with high expression of MMP-9 were lower than those with low expression (both P<0. 05). The binding energies of Sang to MMP-2, MMP-9, and PTGS2 were -8. 8, -8. 6, and -10. 0 kcal/mol, respectively. The mRNA expression of PTGS2, MMP-2, and MMP-9 was lower in the experimental group than that in the control group (all P<0. 05). Conclusions Sang can inhibit the proliferation and migration abilities of bladder cancer T24 cells at certain concentrations without affecting SV-HUC-1 cells. The regulatory effects of Sang on the proliferation and migration abilities of bladder cancer cells may be related to the regulation of MMP-2, MMP-9 and PTGS2 expression and the influence of epithelial cell proliferation-related pathways. [ABSTRACT FROM AUTHOR]