Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tgp22phox), in which overexpression of p22phox was targeted to VSMC. Also, we compared H2O2 generation and expression of specific phenotypic markers of non-migrating with migrating WT versus Tgp22phox VSMC in an in vitro wound scratch model. Enhanced H2O2 production in Tgp22phox versus WT VSMC (p < 0.005) significantly correlated with increased protein content, proliferation, and migration. VSMC migrating across the wound edge produced more H2O2 than non-migrating VSMC (p < 0.05). The expression of synthetic phenotypic markers, tropomyosin 4 and myosin heavy chain embryonic (SMemb), was enhanced significantly, while the expression of contractile marker, smooth muscle alpha-actin, was reduced significantly in migrating versus non-migrating cells, and also in Tgp22phox versus WT (p < 0.005) VSMC. These results are consistent with increased production of ROS accelerating the switch from the contractile to the synthetic phenotype, characterized by increases in proliferation, migration, and expression of TM4 and SMemb and decreased alpha-actin.