© 2022, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.DNA double-strand breaks (DSBs) are commonly appearing deleterious DNA damages, which progressively increase in male germ cells during biological aging. There are two main pathways for repairing DSBs: homologous recombination (HR) and classical nonhomologous end joining (cNHEJ). Knockout and functional studies revealed that, while RAD51 and RPA70 proteins are indispensable for HR-based repair, KU80 and XRCC4 are the key proteins in cNHEJ repair. As is known, γH2AX contributes to these pathways through recruiting repair-related proteins to damaged site. The underlying reasons of increased DSBs in male germ cells during aging are not fully addressed yet. In this study, we aimed to analyze the spatiotemporal expression of the Rad51, Rpa70, Ku80, and Xrcc4 genes in the postnatal mouse testes, classified into young, prepubertal, pubertal, postpubertal, and aged groups according to their reproductive features and histological structures. We found that expression of these genes significantly decreased in the aged group compared with the other groups (P < 0.05). γH2AX staining showed that DSB levels in the germ cells from spermatogonia to elongated spermatids as well as in the Sertoli cells remarkably increased in the aged group (P < 0.05). The RAD51, RPA70, KU80, and XRCC4 protein levels exhibited predominant changes in the germ and Sertoli cells among groups (P < 0.05). These findings suggest that altered expression of the Rad51, Rpa70, Ku80, and Xrcc4 genes in the germ and Sertoli cells may be associated with increasing DSBs during biological aging, which might result in fertility loss.