Androgens are essential for normal spermatogenesis and male fertility, but how androgens exert this effect remains uncertain. Androgen receptors (ARs) are expressed in several testicular cell types, but continuing uncertainty exists over which cell type mediates androgen control of spermatogenesis. Androgen signaling via Sertoli cells (SCs) is essential for complete spermatogenesis, but the role for androgen signaling via peritubular myoid (PTM) cells is contentious. To address this controversy, we generated PTM-specific AR-knockout (PTM-ARKO) mice in which gross reproductive development was normal, but all PTM-ARKO males were azoospermic and infertile. Testis weight was reduced beyond puberty, and in adulthood there was an 86% reduction in germ cells, compared with wild-type littermates. These changes were not explained by any deficits in testosterone, luteinizing hormone, or follicle-stimulating hormone concentrations. SC function was impaired in PTM-ARKO males, indicated by reduced seminiferous tubule fluid production and reduced expression of some androgen-dependent SC genes. Androgen action via PTM cells is therefore essential for normal testis function, spermatogenesis, and fertility in males. This study also provides the first direct evidence for the importance of androgen-driven stromal-epithelial interactions underpinning the regulation of spermatogenesis; PTM-ARKO mice will enable identification of the new molecular pathways involved.—Welsh, M., Saunders, PTK, Atanassova, N., Sharpe, RM, Smith, LB Androgen action via testicular peritubular myoid cells is essential for male fertility.