Radiotherapy can result in temporary or permanent gonadal toxicity in male cancer patients despite the high precision and accuracy of modern radiation treatment techniques. Previous radiobiological studies have shown an effective tissue-sparing response in various tissue types and species following exposure to spatially fractionated radiation. In the present study, we used an ex vivo mouse testicular tissue culture model and a conventional X-ray irradiation device to evaluate the tissue-sparing effect (TSE) of spatially fractionated X-rays for the protection of male fertility from radiotherapy-related adverse effects. We revealed a significant TSE for maintaining spermatogenesis in the ex vivo testes model following spatially fractionated X-ray irradiation. Moreover, we experimentally propose a possible mechanism by which the migration of spermatogonial cells, from the non-irradiated areas to the irradiated ones, in irradiated testicular tissue, is essential for the TSE and maintaining spermatogenesis. Therefore, our findings demonstrate that the control of TSE following spatially fractionated X-rays in the testes has a considerable potential for clinical application. Interdisciplinary research will be essential for further expanding the applicability of this method as an approach for the preservation of male fertility during or after radiotherapy.