The aim of this study was to investigate the inhibition of daidzein or/and regular exercise on breast cancer and to reveal the potential biological mechanisms. BALB/c mice pretreated with regular exercise training for 20 days (15 m/min, 60 min/d) were orthotopically transplanted with mouse breast cancer cells (4T1), and then treated with daidzein (145 mg/kg) by gavage for another 22 days. Results showed that exercise or daidzein inhibited tumor growth in mice to a different degree. Particularly, co-treatment with exercise and daidzein showed an obviously synergistic inhibition on the tumor growth (P < 0.01), compared with the tumor control. Further researches indicated that the combination of exercise and daidzein synergistically mobilized and redistributed natural killer cells through upregulating the level of epinephrine and interleukin-6. Moreover, exercise combined with daidzein induces apoptosis in cancer cells via Fas/FasL-initiated mitochondrial apoptosis signaling pathway. These results suggested that regular exercise combined with daidzein may explore a candidate way to prevent and treat the breast cancer.