Abstract Body: Clinical maturation of arteriovenous fistulas (AVF) is largely determined by the adaptive response of the vein to extreme hemodynamic conditions. Histological and bulk transcriptomic analyses of human veins indicate that this is a profound mechanosensitive and inflammatory reparative process, with subtle molecular differences separating maturation and failure. To look for these subtle changes, we analyzed the microanatomy of veins and AVFs that matured and failed using single-cell RNA sequencing (70,281 cells from 20 individuals) and immunofluorescence (IF, n=48 cross-sections). Comparisons of vein and AVF pairs from the same individuals confirmed an increase in area and cell count per layer after AVF creation. However, in all vascular layers, cell density decreased after anastomosis, reflecting an increase in extracellular matrix (ECM) deposition but a slow rate of proliferation. A reduction in density was specifically observed in MYH11+ smooth muscle cells (SMCs) in the intima and media, and CD31+ endothelial cells (ECs) in the lumen. In contrast, ECM-producing myofibroblasts, PDGFRA+ fibroblasts, and CD68+ macrophages maintained their preoperative density after anastomosis. Single-cell expression analyses further highlighted the role of inflammation and ECM remodeling and defined the cell-to-cell interactions responsible for wall repair in AVFs. These were characterized by the expansion of inflammatory and glycosaminoglycan-producing fibroblasts after anastomosis, as well as the activation of wound healing programs in macrophages, myofibroblasts, and ECs. Of interest, these analyses uncovered an enhanced cross-talk among all these cells in AVFs that failed but not in those that matured. Both single cell sequencing and IF stainings confirmed a higher density of infiltrated macrophages, more ACKR3+ ECs, and upregulation of inflammatory factors in myofibroblasts and fibroblasts in association with AVF failure. This study highlights the importance of the inflammatory-ECM remodeling axis in AVF remodeling and identifies the key cellular players for therapeutic targeting to prevent maturation failure.