Flaxseed (FS) is rich in mammalian lignan precursors and alpha-linolenic acid, which have been suggested as having anticancer effects. Previous studies have shown that 10% FS inhibits the growth of human estrogen-dependent breast cancer (MCF-7) in athymic mice, and it enhances the inhibitory effect of tamoxifen (TAM). This study determined whether the effect of FS, alone or in combination with TAM, is dose dependent, and it explored the potential mechanism of action. Ovariectomized athymic mice with estradiol (E2) supplementation (1.7 mg/pellet, 60-day release) and established MCF-7 tumors were treated with basal diet control (0FS), 5% FS (5FS), 10% FS (10FS), and TAM (TAM/ 0FS; 5 mg/pellet, 60-day release), alone or in combination (TAM/ 5FS and TAM/10FS) for 8 weeks. Compared with control, 5FS and 10FS significantly inhibited tumor growth by 26% and 38%, respectively. TAM/0FS had an effect similar to the 10FS. TAM/ 5FS and TAM/10FS, respectively, induced significant 48% and 43% reductions in tumor size compared with 0FS, and 18% and 10% reductions compared with TAM/0FS. The relative uterine weight was significantly lower in all TAM groups compared with the control. The reduction of tumor growth resulted from decreased cell proliferation and increased cell apoptosis. TAM/ 5FS caused a significantly higher expression of estrogen receptor-alpha (ERalpha) compared with 5FS and TAM/0FS, whereas TAM/10FS had a higher ERalpha than 10FS and TAM/0FS. Compared with the control, progesterone receptor (PgR) expression was significantly reduced in all treatment groups, but insulin-like growth factor-1 (IGF-1) expression was reduced only by 10FS, TAM/5FS and TAM/10FS. Tumor cell proliferation was significantly positively associated with expression of PgR and IGF-1 and negatively associated with apoptosis and ERalpha. Apoptosis was only associated with ERalpha. In conclusion, FS inhibited MCF-7 tumor growth in a dose-dependent manner and enhanced the inhibitory effect of TAM due to the modulation of ER and growth factor signal transduction pathways.