AIM: To explore the mechanism of berberine on breast cancer cells based on network pharmacology and in vitro cell experiments. METHODS: Firstly, berberine and breast cancer were taken as the research objects, the intersection targets of the two were screened by VEEN diagram, GO function and KEGG enrichment analysis were performed by R language, and molecular docking and visualization were carried out by Autodock Vina and Pymol software. Then, berberine treated breast cancer MCF-7 cells for 24 h, and then in vitro cell experiments were performed. CCK-8 was used to detect cell viability, Edu and plate cloning were used to detect cell proliferation and cloning, and apoptosis was detected by An-nexin V-FITC/PI double staining and Western blot. Laser confocal and CETSA were used to verify the binding effect of berberine and AKT1 protein. RESULTS: The results of network pharmacology showed that berberine had a good binding to the core targets AKT1, AKT2 and MAPK3. Berberine (20, 40, 80 μmol/L) significantly inhibited the proliferation and cloning ability of MCF-7 cells in a concentration-dependent manner (P<0.05, P<0.01). The results of laser confocal and CETSA experiments showed that berberine and AKT1 had a binding effect, and the stability of the two was enhanced after the combination. CONCLUSION: Berberine inhibits MCF-7 cell proliferation and induces apoptosis in human breast cancer cells by targeting binding to AKT1 protein.