ATP6AP2 is a multi-functional protein, involved in a number of cellular pathways including WNT and MAPK signalling, protein sorting and folding, and receptor-mediated endocytosis and recycling. Many of these functions depend on its interaction with the vacuolar H+-ATPase (V-ATPase). Due to its role in signalling pathways, we hypothesised that ATP6AP2 plays a role in gonad development. To test this hypothesis, we deleted Atp6ap2 specifically in gonadal somatic cells during foetal development using the Nr5a1-Cre mouse. Surprisingly, these mice appear to develop normally pre-birth, and were born at the expected Mendelian ratio. However, both males and females were infertile. At three months of age conditional Atp6ap2-deficient XY mice presented with significantly smaller testes caused by the loss of spermatogenic cells. Subsequent analysis demonstrated that the loss of Atp6ap2 in testicular somatic cells resulted in the loss of round spermatids by apoptosis, likely caused by a disruption of cell-cell communication due to defects in the trafficking of junctional proteins. The loss of spermatids led to increased differentiation and proliferation of progenitor cells, ultimately leading to exhaustion of the stem and progenitor pool and hence Sertoli cell-only tubule. Together this data suggests ATP6AP2 as a novel candidate for male infertility in humans.