S-phase kinase-associated protein-2 (Skp2) plays a key role in ubiquitin-mediated proteolysis,resulting in the progression of cells from a quiescent to a proliferative state. Skp2 is overexpressed in a variety of tumors and inversely correlated with p27 expression, including breast cancer. In this study, we used small interfering RNAs (siRNAs) to inhibit Skp2 expression in human breast cancer MCF-7 cells and thereby investigate if knockdown of Skp2 by RNA interference (RNAi) would inhibit breast cancer cell growth and influence the effects of epirubicin on MCF-7 cells. Three Skp2 siRNA constructs were recombined and transiently transfected human breast cancer MCF-7 cells. Down-regulation of Skp2 was confirmed by reverse transcription-polymerase chain reaction and Weste blot analysis. We showed that Skp2 siRNA transfection decreased Skp2 protein and induced the accumulation of p27 protein in MCF-7 cells. Skp2 siRNA inhibited the cell proliferation both in vitro and in vivo. Knockdown of Skp2 by RNAi increased cellular apoptosis in vitro. Treatment with Skp2 siRNA followed by treatment with epirubicin further inhibited the proliferation of cancer cell lines, compared with control siRNA followed by epirubicin. Therefore,knockdown of Skp2 expression by RNAi inhibits breast cancer cell growth and enhances the effect of epirubicin, siRNA-mediated gene silencing of Skp2 could be a novel cancer gene therapy for the suppression of p27 down regulation.