The migration of vascular smooth muscle cells (SMC) and fibroblasts into the intima after vascular injury is a central process in vascular lesion formation. The elevation of transmural interstitial flow is also observed after damage to the vascular endothelium. We have shown previously that interstitial flow upregulates matrix metalloproteinase-1 (MMP-1) expression which in turn promotes SMC and fibroblast migration in collagen I gels. In this study, we investigated further the mechanism of flow-induced MMP-1 expression. An ERK1/2 inhibitor (PD-98059) completely abolished interstitial flow-induced SMC migration and MMP-1 expression. Interstitial flow promoted ERK1/2 phosphorylation while PD-98059 abolished flow-induced activation. Silencing ERK1/2 completely abolished MMP-1 expression and SMC migration. In addition, interstitial flow increased the expression of AP-1 transcription factors (c-Jun and c-Fos), while PD-98059 attenuated flow-induced expression. Knocking down c-jun completely abolished flow-induced MMP-1 expression, while silencing c-fos did not affect MMP-1 expression. Taken together, our data indicate that interstitial flow induces MMP-1 expression and SMC migration in collagen I gels via an ERK1/2-dependent and c-Jun-mediated mechanism and suggests that interstitial flow, ERK1/2 MAPK, c-Jun, and MMP-1 may play important roles in SMC migration and neointima formation after vascular injury.