Aim To study the role and mechanism of dehydroepiandrosterone (DHEA) in the proliferation of vascular smooth muscle cells (VSMCs) induced by angiotensin Ⅱ (AngⅡ). Methods VSMCs were cultured with AngⅡ in vitro, then treated with DHEA. The effect of DHEA on the proliferation of VSMCs induced by AngⅡ was detected by MTS and cell counting. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the effects of DHEA on the expressions of proliferating cell nuclear antigen (PCNA), Krüppel-like factor 5 (KLF5) and inducible nitric oxide synthase (iNOS) in AngⅡ-induced VSMCs. The effect of DHEA on the concentration of peroxynitrite anion (ONOO－) in the medium of AngⅡ-induced VSMCs was examined by enzyme-linked immunosorbent assay (ELISA). Endogenous iNOS was knocked down by transfecting VSMCs with iNOS-specific siRNA (si-iNOS) or nonspecific siRNA (si-Ctrl), and then treated the cells with or without DHEA; Western blot was performed to examine the expression of PCNA. Results The results of MTS and cell counting showed that DHEA could significantly inhibit the proliferation of VSMCs induced by AngⅡ (P＜0.05). The results of qRT-PCR and Western blot showed that DHEA significantly inhibited the expressions of PCNA, KLF5 and iNOS in VSMCs induced by AngⅡ at the level of protein and mRNA (P＜0.05). ELISA results showed that DHEA could reduce the concentration of ONOO－ in culture medium of VSMCs treated with AngⅡ (P＜0.05). Western blot results suggested that DHEA had no effect on AngⅡ-induced PCNA protein expression after knocked down endogenous iNOS (P＞0.05). Conclusion DHEA may inhibit the proliferation of VSMCs induced by AngⅡ probably through down-regulation of KLF5 expression by inhibiting ONOO－ produced by iNOS.