Platelet-derived growth factor (PDGF) is released from vascular smooth muscle cell (VSMC), after percutaneous coronary intervention and is related with neointimal proliferation and restenosis. Adrenal steroid dehydroepiandrosterone sulfate (DHEAS), the sulfated prohormone of dehydroepiandrosterone has shown remarkable biological activity against proliferation of VSMC in some animal and clinical studies. Combinations of DHEAS with other agents have also shown promising results, with acquiring more efficient effect. Berberine is a naturally occurring isoquinoline alkaloid. To investigate their effects in combination, a VSMC cell line A7r5 was stimulated by PDGF-BB (dimer of the B chain of PDGF), and then treated with berberine and/or DHEAS in the current study. Cell proliferation assay, cell cycle assay, Western blot, and co-immunoprecipitation were analyzed in A7r5 cells. Antiproliferative effects of berberine and/or DHEAS targeting the Skp2/p27 pathways were evaluated. Berberine and DHEAS can both inhibit the growth of A7r5 cells. Berberine induces cell cycle arrest and potentiates the inhibitory effect of DHEAS through disrupting the binding of p27, p21 with Skp2. Berberine and DHEAS decreased the expression of CDK2, CDK4, PCNA, cyclin D1, and cyclin E, which was induced by PDGF-BB. Being treated with berberine and DHEAS also promoted p27 and p21 bind to CDK2, so the proliferation of A7r5 cells induced by PDGF-BB was inhibited. The data provide evidence that berberine acts through the inhibition of p27-Skp2 and p21-Skp2 with subsequent activation of the cell cycle arrest, which leads to the increase in sensitivity to DHEAS. In summary, the findings suggest that combined berberine and DHEAS will be active in the prevention of restenosis after angioplasty treatment, and the treatment of atherosclerosis.