Certain fatty acids in canola oil (CAN) have been associated with a reduced risk of breast cancer. This study assessed the effects of CAN on proliferation and death of human breast cancer cells in vitro and in vivo in chemically induced mammary carcinogenesis. We hypothesize that CAN reduces breast cancer cell growth by inducing cell death. In a series of in vitro experiments, human breast cancer T47D and MCF-7 cells were cultured and treated with CAN and two chemotherapeutic drugs, tamoxifen and cerulenin. Cell proliferation and caspase-3 and p53 activities were measured. Reduced cancer cell growth and increased expression of caspase-3 and p53 were seen in T47D and MCF-7 cells treated with CAN. Moreover, CAN showed synergistic cancer cell growth inhibition effects with tamoxifen and cerulenin. In a subsequent live animal experiment, 42 female Sprague-Dawley rats were randomly assigned to corn oil (CORN) or CAN diets, and mammary tumors were chemically induced by N-nitroso-N-methylurea. CAN-dieted rats had reduced tumor volumes and showed an increased survival rate as compared to CORN-dieted rats. We demonstrated that CAN has suppressive effects on cancer growth, and reduces tumor volumes. The results suggest that CAN may have inhibitory effects on breast cancer cell growth, and warrants further investigation of the synergistic effects of CAN with anti-cancer drugs.