Abstract Body: Background: Arteriovenous fistulae (AVF) are the preferred surgical procedure for vascular access. However, 60% of AVF fail within one year due to aggressive neointimal hyperplasia, which significantly increases morbidity and mortality. Matrix metalloproteinases-3 (MMP-3), also known as stromelysin-1, is a member of the metalloproteinase family that plays a critical role in the pathogenesis of many human disorders by degrading extracellular matrix and regulating molecular signaling pathways. However, the role of MMP-3 in AVF neointimal failure has not been explored. Methods and Results: We observed that MMP-3 was time-dependently induced by FBS and the growth factor PDGF-BB in cultured venous SMCs. MMP-3 was also highly expressed in the venous neointimal SMCs in an AVF rodent model, as well as in human AVF specimens. Knockdown of MMP-3 significantly suppressed venous SMC proliferation, whereas overexpression of MMP-3 facilitated cell growth in vitro. Importantly, deficiency of global and SMC-specific MMP-3 significantly reduced neointimal hyperplasia and improved patency after AVF creation. Mechanistic studies showed that MMP-3-mediated SMC proliferation and AVF neointimal hyperplasia were regulated via the FAK-AKT signaling pathway. Conclusion: These data suggest that MMP-3 is a key mediator of AVF neointimal failure. Targeting MMP-3 may be a novel therapeutic strategy to prevent AVF neointimal failure and improve overcomes in patients requiring hemodialysis.