In this study, we evaluated the anti-tumor potential of luteolin (30mg/kg, p.o.), combined with cyclophosphamide (10mg/kg, i.p.) (LU+CYC) orally administered for 20 days; and CYC individually for 10 days against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in Wistar rats. Combination treatment (LU+CYC) inhibited the incidence rate of tumors and decreased tumor volume significantly without changing the total body weight of the animals. Long-term treatment did not show any apparent toxicity in rats. The CYC-treated group showed potential reduction of tumor volume (74%), severe toxicity, and loss of body weight. In order to elucidate the anticancer mechanism of luteolin, antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) generation in the liver, kidney and breast, as well as protein profiles, were also examined. Biochemical analysis of the combination-treated group showed significant (P<0.01; P<0.05) inhibition of lipid peroxide (LPx) formation (oxygen-free radicals), the level and the activity of SOD, CAT and GPx were found to be very high than the LU and CYC individually treated rats at a 30mg/kg dose. 2D gel electrophoresis analysis revealed that (56kDa) high molecular weight protein was detected in tumors of rats receiving combination treatment than the cancer controls. The biological significance of that protein involved for the dysfunction of cancer cells and induces apoptosis. Histopathological changes also confirmed the formation of tumor tubules and neovascularization after the treatment. Overall, these results suggest that the combination treatment provided antioxidant defense with strong chemopreventive activity against the genesis of DMBA-induced mammary tumors.