Neointima formation often occurs in regions where the endothelium has been damaged and the transmural interstitial flow is elevated. Vascular smooth muscle cells (SMC) and fibroblasts/myofibroblasts (FB/MFB) contribute to intimal thickening by migrating from the media and adventitia into the site of injury. In this study, for the first time, the direct effects of interstitial flow on SMC and FB/MFB migration were investigated in an in vitro 3-dimensional (3-D) system. Collagen I gels were used to mimic 3-D extracellular matrix (ECM) for rat aortic SMC and FB/MFB. Exposure to interstitial flow-induced by 1 cmH₂O pressure differential (shear stress ~0.05 dyn/cm², flow velocity ~0.5 µm/s, and Darcy permeability ~10^-11 cm²) substantially enhanced cell motility. Matrix metalloproteinase (MMP) inhibitor (GM 6001) abolished flow-induced migration augmentation, which suggested that enhanced motility was MMP-dependent. Upregulation of MMP-1 played a critical role for flow-enhanced motility, which was further confirmed by silencing MMP-1 gene expression. Longer exposures to higher flows suppressed the number of migrated cells, although MMP-1 gene expression remained high. This suppression was a result of both flow-induced tissue inhibitor of metalloproteinase-1 (TIMP-1) upregulation and increased apoptotic and necrotic cell death. Interstitial flow did not affect MMP-2 gene expression or activity in the collagen I gel for any cell type. Our findings shed light on the mechanism by which vascular SMC and FB/MFB contribute to intimal thickening in regions of vascular injury where interstitial flow is elevated.