NADPH oxidase-derived reactive oxygen species (ROS) contribute to atherosclerosis. We have previously shown that smooth muscle cells isolated from atherosclerotic lesions of murine aorta (plaque-derived SMC) exhibit increased expression of the NADPH oxidase subunit Nox4, increased ROS levels, and decreased rate of growth, as compared to medial-derived SMC. In this study, we tested the hypothesis that increased expression of Nox4 contributes to growth arrest in plaque-derived SMC. Sequence-specific gene silencing of Nox4 by RNA interference (siNox4) reduced mRNA expression of Nox4 by 80% and decreased superoxide levels by 36% in plaque-derived SMC, as compared to cells treated with control siRNA. Cell growth of plaque-derived SMC, however, was not improved following siNox4 treatment. To further investigate the effect of Nox4 on SMC growth, human aortic SMC were transfected with pCMVNox4 (Nox-SMC) or pCMVscript (control-SMC). Real-time PCR confirmed a 6-fold increase of Nox4 expression, whereas chemiluminescence indicated a 2-fold increase in NAPDH oxidase activity, in Nox-SMC compared to control-SMC. In 10% serum, Nox-SMC exhibited increased percentage of cells in G0/G1 phase. As detected by Western blotting, phosphorylation of ERK and Akt were increased in plaque-derived SMC compared to medial SMC. siNox4 failed to reduce phosphorylation of Akt or ERK in plaque-derived SMC. Forty-eight hour treatment of plaque-derived SMC, but not medial SMC, with wortmannin (10 μM), an inhibitor of PI3k/Akt activation, or PD98059 (20 μM), an inhibitor of ERK activation, increased the percentage of cells entering S phase. In summary, (1) reduction of Nox4 expression by siRNA lowers ROS levels but does not improve growth of plaque-derived SMC. (2) Expression of Nox4 increases ROS levels and decreases proliferation of human SMC. (3) Activation of Akt and ERK is increased in plaque-derived SMC, and pharmacologic inhibition of activation induces proliferation. In conclusion, reduction in Nox4-derived ROS is not sufficient to improve growth of plaque-derived SMC.