Background Interstitial flow directly affects cells that reside in tissues and regulates tissue physiology and pathology by modulating important cellular processes including proliferation, differentiation, and migration. However, the structures that cells utilize to sense interstitial flow in a 3-dimensional (3D) environment have not yet been elucidated. Previously, we have shown that interstitial flow upregulates matrix metalloproteinase (MMP) expression in rat vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts via activation of an ERK1/2-c-Jun pathway, which in turn promotes cell migration in collagen. Herein, we focused on uncovering the flow-induced mechanotransduction mechanism in 3D. Methodology/Principal Findings Cleavage of rat vascular SMC surface glycocalyx heparan sulfate (HS) chains from proteoglycan (PG) core proteins by heparinase or disruption of HS biosynthesis by silencing N-deacetylase/N-sulfotransferase 1 (NDST1) suppressed interstitial flow-induced ERK1/2 activation, interstitial collagenase (MMP-13) expression, and SMC motility in 3D collagen. Inhibition or knockdown of focal adhesion kinase (FAK) also attenuated or blocked flow-induced ERK1/2 activation, MMP-13 expression, and cell motility. Interstitial flow induced FAK phosphorylation at Tyr925, and this activation was blocked when heparan sulfate proteoglycans (HSPGs) were disrupted. These data suggest that HSPGs mediate interstitial flow-induced mechanotransduction through FAK-ERK. In addition, we show that integrins are crucial for mechanotransduction through HSPGs as they mediate cell spreading and maintain cytoskeletal rigidity. Conclusions/Significance We propose a conceptual mechanotransduction model wherein cell surface glycocalyx HSPGs, in the presence of integrin-mediated cell-matrix adhesions and cytoskeleton organization, sense interstitial flow and activate the FAK-ERK signaling axis, leading to upregulation of MMP expression and cell motility in 3D. This is the first study to describe a flow-induced mechanotransduction mechanism via HSPG-mediated FAK activation in 3D. This study will be of interest in understanding the flow-related mechanobiology in vascular lesion formation, tissue morphogenesis, cancer cell metastasis, and stem cell differentiation in 3D, and also has implications in tissue engineering.