Canine mammary gland tumors are the most frequent cause of cancer in female dogs. Numerous studies using cancer cell lines and clinical trials have indicated that various natural products and antioxidants reduce or possibly prevent the development of cancer. Berberine (BBR), the most important alkaloid in the Berberidaceae, which exerts a wide range of pharmacological and biochemical effects, has drawn much attention due to its particularly high antitumor activity in vitro and in animal studies. The aim of the present study was to investigate the antiproliferative effect of BBR against a canine mammary gland carcinoma cell line (CF41.Mg) in vitro. CF41.Mg cells were cultured in RPMI-1640 medium containing 10% heat inactived fetal bovine serum (FBS) and 100 mg/ml peniciline-streptomycin. Subsequently the cells were treated with different concentrations of BBR chloride (10, 25, 50, 100 and 200 µM) at a density of 12,000 cells/well in 96-well plates. Following treatment, the MTT assay was used to detect cell viability after 24-, 48- and 72-h incubations at 37°C with 5% CO2. The results indicated that BBR inhibited proliferation of canine mammary gland carcinoma cells, as treatment with 100 µM BBR for 24 h resulted in a significant decrease in cell viability (P<0.005). As the present study demonstrated that BBR (10-200 µM) induced cancer cell death, it is proposed that BBR may serve as a candidate agent against canine mammary tumor cells via its antiproliferative activity.