Deteriorating physical function in ageing men is postulated to involve reduced testosterone levels and is estimated to affect 6-39% of men over 40 and up to 50% of men over 85. Although age-related reductions in Leydig cell steroidogenic capacity and altered hypothalamic-pituitary-gonadal (HPG) communication have been described, little is known about mechanisms triggering these changes. Inhibin is produced predominantly in the testis and circulating inhibin-B concentrations decline from ~30 years. Inhibin is a potent activin inhibitor, hence reduced inhibin implicates upregulated activin signalling in age-related testicular decline. We used mice lacking one copy of the inhibin alpha gene to investigate the consequences of an altered inhibin:activin ratio on testicular ageing. Testicular and circulating inhibin-α subunit concentrations were 30% lower in Inha+/- mice compared to wildtype, but activin A, activin B, follicle stimulating hormone and testosterone concentrations were not different (n>6). Inha+/- mice displayed an age-related decline in daily sperm production between 8 and 26 weeks (-27%, P<0.05), whereas sperm production in wildtype mice increased during this period (+31%, P<0.01). Flow cytometric ploidy analysis confirmed that Inha+/- mice had a significantly lower testicular haploid cell content compared to wildtype, correlating with somatic cell changes. Sertoli cell flow cytometry identified ~30% of Sertoli cells in S-phase in both Inha+/+ and Inha+/- mice at 8 weeks. By 26 weeks, the proportion of S-phase Inha+/+ Sertoli cells had declined to 19% (P<0.001) but remained elevated in Inha+/- mice (35.8%). Testosterone is necessary for optimal spermatogenesis and for Sertoli cell terminal differentiation and cell cycle quiescence. Immunohistochemical analysis of steroidogenic enzymes identified that expression of CYP11A1 (p450scc), the rate-limiting enzyme upstream of testosterone synthesis, was reduced in some Inha+/- Leydig cells at 26 weeks. Suggestive of focal regions of androgen deficiency, these findings correlate inhibin insufficiency with Leydig cell failure and age-related testicular decline.