Background/Objective Arteriovenous fistulas (AVFs) are the preferred vascular access for hemodialysis of patients with end-stage renal disease. However, there is a high incidence of AVF failures caused by insufficient outward remodeling or venous neo-intimal hyperplasia formation. Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) play an important role in many cardiovascular diseases. Abnormal VSMC proliferation and migration could be abolished by inhibition of mitochondrial division. Method We found that abnormal proliferation and migration of VSMCs and increased mitochondrial fission were associated with AVF stenosis in patients. We also investigated the mechanisms, particularly the role of mitochondrial dynamics, underlying these VSMC behaviors. In vitro, we observed that inhibition of mitochondrial fission and Akt phosphorylation can diminish proliferation and migration of VSMCs induced by platelet-derived growth factor-BB (PDGF-BB). In vivo, daily intraperitoneal injections of mitochondrial division inhibitor 1 (Mdivi-1) decreased VSMC proliferation and reduced AVF wall thickness in a rat AVF model. Conclusion and Result Our results suggest that inhibition of mitochondrial fission improves AVF patency by reducing wall thickening through the PI3K/Akt signaling pathway. Therefore, inhibition of mitochondrial fission has the clinical potential to improve AVF patency.