Nitric oxide (NO) is produced via oxidation of l-arginine by nitric oxide synthases (NOSs), and is known as inducible (iNOS), neuronal, endothelial or testis-specific. Suggesting important functions for NOS in the normal rat and mouse testis, iNOS is reported to be constitutively expressed in Leydig cells (LC), Sertoli cells (SC) and germ cells. In our study, we sought to provide further insights into the roles of iNOS in the adult mouse testis using iNOS-/- mice. Perfusion-fixed testes from wild type (WT) and iNOS-/- mice were used for histological and stereological evaluations. Some of the mice had been injected with 3H-thymidine to label proliferating cells and to determine the duration of spermatogenesis that was unaffected in iNOS-/- mice. Both LC nuclear volume and individual cell size were significantly decreased in iNOS-/- mice, but the total number of LC per testis was increased (p < 0.05) by approximately 16%. The number of SC per testis was strikingly increased (approximately twofold) in iNOS-/- mice, and testis weight and DSP per gram of testis (spermatogenic efficiency) were similarly increased. The anogenital distance was also significantly increased in iNOS-/- mice, and this key endpoint suggests that the augmentation observed for the SC number may be related to increased foetal T-exposure during the masculinization programming window. Compared with WT testes, the numbers of spermatocytes and spermatids and SC per tubule cross sections were significantly increased in iNOS-/- mice. Except for stages V–VI and VII–VIII, iNOS-/- mice exhibited approximately 3.5-fold fewer apoptotic germ cells than in WT mice. Taken together, our results provide new evidence that iNOS plays an important role in numerical and functional regulation of key somatic cells in the testis, which in turn impacts on germ cells and their survival and thus on daily sperm production.