BACKGROUND: The neuroretinal vascular system is comprised of three interconnected layers. The initial superficial vascular plexus formation is guided by astrocytes around birth in mice. The formation of the deep and intermediate vascular plexuses occurs in the second postnatal week and is driven by Müller-cell-derived angiogenic signaling. Previously, we reported that microglia play an important role in regulating astrocyte density during superficial vascular plexus formation. Here, we investigated the role of microglia in regulating Müller-cell-dependent inner retinal vascular development. METHODOLOGY: In this study, we depleted microglia during retinal development using Csf1R antagonist (PLX5622). We characterized the developmental progression of inner retinal vascular growth, effect of microglial depletion on inner retinal vascular growth and Müller cell marker expressions by immunostaining. Differential expressions of genes in the control and microglia depleted groups were analyzed by mRNA-seq and qPCR. Unpaired t-test was performed to determine the statistical differences between groups. RESULTS: This study show that microglia interact with Müller cells and the growing inner retinal vasculature. Depletion of microglia resulted in reduced inner retinal vascular layers densities and decreased Vegfa isoforms transcript levels. RNA-seq analysis further revealed that microglial depletion significantly reduced specific Müller cell maturation markers including glutamine synthetase, responsible for glutamine biosynthesis, necessary for angiogenesis. CONCLUSIONS: Our study reveals an important role for microglia in facilitating inner retinal angiogenesis and Müller cell maturation.