Breast cancer is one of the most lethal cancers affecting women worldwide and was estimated to account for ~30% of all new cancer diagnoses in women. Although available evidence has proved the tumor suppressor role of genistein in cancer, the underling mechanisms have remained to be fully elucidated. S-phase kinase-associated protein 2 (Skp2) has been revealed to critically enhance the pathogenesis of multiple human cancers. The present study determined whether genistein exerts its anti-tumor function by suppressing Skp2 in breast cancer cells. Genistein significantly inhibited the proliferation, invasion and migration of breast cancer cells. Furthermore, genistein treatment also induced marked apoptosis and a typical cell cycle arrest in G2/M phase. Mechanistically, genistein treatment was identified to cause a significant downregulation of Skp2. Two crucial tumor suppressors, p21 and p27, were upregulated in genistein-treated breast cancer cells. The present results revealed that genistein exerted its tumor suppressor effect at least partially via inhibition of Skp2 and promotion of its downstream targets p21 and p27. Therefore, inactivation of Skp2 by genistein may be a promising approach for breast cancer treatment.