Spaceflight experiments carried out in microgravity environments have revealed that exposure to altered gravity condition results in alteration of several cellular functions and, consequently, of several apparatuses. There is some evidence in the literature indicating that spaceflight affects the physiology of the testis. The data on effects of spaceflight or simulated microgravity on testicular function, however, sometimes appear contradictory. In the present study we used an in vitro experimental model in order to investigate the direct effects of microgravity on testicular tissue. We generated a microgravity environment using the Rotating Wall Vessel and performed experiments on testicular fragments isolated from pre-pubertal rats. In this model we then analyzed several parameters such as histological integrity, cell proliferation, cell apoptosis, occludin distribution pattern, and hormonal secretions. The emerging picture shows some alterations of testicular tissue physiology. Interestingly, we also demonstrate for the first time that, in organ culture, Leydig cell survival is severely affected by simulated microgravity.