The proton pumping V-ATPase drives essential biological processes, such as acidification of intracellular organelles. Critically, the V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting kidney intercalated cells (ICs). However, little is known about the molecular regulation of V-ATPase in mammals. We identified a novel interactor of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 (Dmxl1), aka Rabconnectin-3A. The yeast homologue of Dmxl1, Rav1p, is part of a complex that catalyzes the reversible assembly of the domains. We, therefore,hypothesized that Dmxl1 is a mammalian V-ATPase assembly factor. Here, we generated kidney IC-specific Dmxl1 knockout (KO) mice, which had high urine pH, like B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting showed decreased B1 expression and B1 (V1) and a4 (VO) subunits were more intracellular and less colocalized in Dmxl1 KO ICs. In parallel, subcellular fractionation revealed less V1 associated B1 in the membrane fraction of KO cells relative to the cytosol. Furthermore, a proximity ligation assay performed using probes against B1 and a4 V-ATPase subunits also revealed decreased association. We propose that loss of Dmxl1 reduces V-ATPase holoenzyme assembly, thereby inhibiting proton pumping function. Dmxl1 may recruit the V1 domain to the membrane and facilitate assembly with the VO domain and in its absence V1 may be targeted for degradation. We conclude that Dmxl1 is a bona fide mammalian V-ATPase assembly factor.