The International Space Station will allow extended habitation in space and long-term exposure to microgravity. A concern is the impact of long-term microgravity exposure on the ability of species to reproduce. The model often used to simulate microgravity is rat hindlimb suspension (HLS) where the hindlimbs are elevated above the cage floor with a tail harness. Experiments described here are the first to examine the effect of long-term HLS on testicular function in adult male rats. Free roaming (controls), tail-only (TO) harnessed but with hindlimbs in contact with the cage floor, and HLS animals were tested for 6 weeks. Cryptorchidism was prevented in TO and HLS animals by partially constricting the inguinal canal with sutures. All parameters were compared at the end of the 6-week experiment. Testicular weights and spermatogenesis were significantly reduced by HLS such that no spermatogenic cells beyond round spermatids were present and the epididymides were devoid of mature sperm. In many tubules, loss of all germ cells except a few spermatogonia resulting in histopathology similar to Sertoli cell only was observed. Control and TO spermatogenesis appeared unaffected. Sertoli and Leydig cell appearance, and testosterone, LH, FSH, epididymal weight, and seminal vesicle weight were unchanged by HLS. Cortisone was not elevated by HLS, thus stress may not be a factor. These results demonstrate that spermatogenesis is severely inhibited by long-term HLS, while testicular androgen production is not. These results have significant implications regarding serious effects of long-term exposure to microgravity on the reproductive capability of scrotal mammals, including man.