Canola oil (CAN), considered to be a well balanced omega-6/omega-3-containing oil, has a very low level of saturated fat (7%) and relatively high levels of oleic acid (62%) and omega-3 fatty acids (11%). The beneficial effects of alpha-linolenic and oleic acids in CAN are widely accepted in human health, particularly in cardiovascular disease, but limited data are available regarding cancer, including breast cancer. We postulated that due to its low omega-6 to omega-3 fatty acid ratio (2:1) and high oleic acid content, a CAN-supplemented diet might alter tumor cell membrane fluidity. This CAN-mediated change in membrane fluidity may result in the inhibition of the binding of estrogen to receptors on the plasma membrane, which in turn may bring about a change in cell signaling, which might lead to reduced tumor cell growth. Secondly, we hypothesized that CAN, due to its fatty acid composition, may sensitize drug resistant breast cancer cells to drug treatment by altering tumor cell membrane fluidity. This change in membrane fluidity may result in suppressed expression of the membrane-associated transport protein, P-glycoprotein, and in turn, increase the intracellular accumulation of the anticancer drug Doxorubicin (DOX), thereby enhancing its cancer-killing activity. The specific objectives of the first postulation were to determine if dietary CAN 1) reduces susceptibility to chemically-induced mammary carcinogenesis in vivo, and 2) affects proliferation and apoptosis of human breast cancer cells in vitro. The specific aims of the second hypothesis were to determine if dietary CAN in combination with DOX 1) reduces susceptibility to drug resistant human breast cancer in vivo, and 2) affects proliferation and apoptosis in drug resistant human breast cancer cells in vitro . To study these hypotheses, we investigated the effect of dietary CAN on the development and growth of chemically-induced carcinogenesis in Sprague-Dawley rats. Secondly, we established a correlation between 1 mM CAN treatment and inhibition of growth of human breast cancer cells (using estrogen positive MCF-7 and T47D human breast cancer cells) in culture. The third experiment examined the effect of dietary CAN on the growth of DOX-resistant human breast cancer cells (MCP-7/DOX) in vitro and in nude mice, Cell proliferation as well as caspase-3 and p53 tumor suppressor protein and tumor cell membrane fluidity were measured. Control (CON) cells were treated with ethanol at appropriate concentration. Results revealed that the CAN supplemented diet reduced tumor incidence in rats as compared to the CON group. Mammary tumor volumes were significantly reduced while the survival rates were increased in the CAN-fed group compared with the CON-fed group. In vitro, CAN significantly inhibited growth of both T47D and MCF7 cancer cells. In addition, CAN plus the drugs tamoxifen or cerulenin further suppressed growth of cancer cells as compared to tamoxifen or cerulenin alone. Caspase-3, p53, and cell death were also increased by CAN treatment. Also, CAN treatment significantly reduced growth of drug resistant human breast cancer cells in culture and in nude mice. DOX plus CAN treatment further inhibited growth and increased caspase-3 activity of MCF-7/DOX cells as compared to CON or DOX alone. Tumor cell membrane fluidity was significantly higher in the CAN-treated MCF-7/DOX cells as compared to the CON. Our data suggest that canola oil has inhibitory effects on breast cancer growth and drug resistance and warrants further investigation of the detailed mechanistic events responsible for this phenomenon.