Microglia, as the resident immune cells of the central nervous system (CNS), maintain neural homeostasis by orchestrating the clearance of necrotic neurons, cellular debris, and pathogens through phagocytosis. This intracellular process involves a dynamic cascade of protein-mediated events, including cargo recognition and phagosome formation, phagosome maturation, and lysosomal degradation. Phagosome maturation critically relies on V-ATPase-driven acidification, with proton transport efficiency predominantly determined by its a subunit. In mammals, the V-ATPase a subunit has four isoforms (a1, a2, a3 and a4), whereas zebrafish (Danio rerio) possess only the three isoforms (a1, a2 and a3), with marked differences observed in their targeted cell types and suborganellar localization patterns. Notably, the V-ATPase a3 subunit is primarily localized on osteoclast lysosomes, facilitating bone resorption by acidification. The preliminary studies have revealed its conserved lysosomal localization in zebrafish microglia; however, its functional role in microglial development and regulatory mechanisms underlying phagosome maturation remain elusive. To address this knowledge gap, we employed a V-ATPase a3 subunit-deficient (tcirg1b^−/−) zebrafish model, integrating whole-mount in situ hybridization, immunofluorescence, co-immunoprecipitation (Co-IP), and apoptosis assays. Key findings include: The a3 subunit is expressed during early zebrafish development; a3 subunit deficiency caused abnormal phagosome accumulation, microglial swelling and decreased activity, showing an “indigestion-like” phenotype; By exogenous labeling of late phagosomes and immunofluorescence staining, it was confirmed that the defect of V-ATPase a3 subunit impaired late phagosome-lysosome fusion in microglia; Co-IP demonstrated direct binding between the a3 subunit and Rab7. Rab7 knockdown phenocopied the “indigestion” morphology, suggesting a cooperative role in phagosome degradation. These results demonstrate that the V-ATPase a3 subunit regulates microglial phagosome maturation by mediating Rab7-dependent phagolysosomal fusion. In conclusion, this study not only elucidates the molecular mechanism underlying microglial phagocytic capacity but also provides novel insights into the evolutionarily conserved roles of V-ATPase isoforms in cellular clearance pathways.