Among all tissues and organs, the mammary gland is unique because most of its development occurs in adulthood. Notch signaling has a major role in mammary gland development and has been implicated in breast cancer. The vacuolar-ATPase (V-ATPase) is a proton pump responsible for the regulation and control of pH in intracellular vesicles and the extracellular milieu. We have previously reported that a2V-ATPase (a2V), an isoform of ‘a' subunit of V-ATPase, regulates processing of Notch receptor and alters Notch signaling in breast cancer. To study the role of a2V in mammary gland development, we generated an a2V-KO model (conditional mammary knockout a2V mouse strain). During normal mammary gland development, the basal level expression of a2V increased from puberty, virginity, and pregnancy through the lactation stage and then decreased during involution. Litters of a2V-KO mice weighed significantly less when compared with litters from wild-type mice and showed reduced expression of the lactation marker β-casein. Whole-mount analysis of mammary glands demonstrated impaired ductal elongation and bifurcation in a2V-KO mice. Consequently, we found disintegrated mammary epithelium as seen by basal and luminal epithelial staining, although the rate of proliferation remained unchanged. Delayed mammary morphogenesis in a2V-KO mice was associated with aberrant activation of Notch and TGF-β (transforming growth factor-β) pathways. Notably, Hey1 (hairy/enhancer-of-split related with YRPW motif) and Smad2, the key downstream mediators of Notch and TGF-β pathways, respectively, were upregulated in a2V-KO mice and also in human mammary epithelial cells treated with a2V siRNA. Taken together, our results show that a2V deficiency disrupts the endolysosomal route in Notch and TGF signaling, thereby impairing mammary gland development. Our findings have broader implications in developmental and oncogenic cellular environments where V-ATPase, Notch and TGF-β are crucial for cell survival.