Background: The present study aimed to investigate the mechanism of low-dose ionizing radiation (IR) induced apoptosis of undifferentiated spermatogonia in vivo and in vitro. Methods: Following 50 mGy IR, testicular tissues were collected from the adult DBA/2 mice at 1, 2 and 24 h; mice in the control group received pseudo-irradiation. Immunofluorescence (IF) staining and TUNEL were performed to assess DNA damage and apoptosis, respectively, in the irradiated testicular tissues. Furthermore, the spermatogonia were also irradiated in vitro, and the expression of apoptosis-related proteins was detected by Western blotting. TUNEL and flow cytometry were applied to assess cell apoptosis. Results:.H2AX ( a marker of DNA damage) was up-regulated in the seminiferous tubules at 1 and 2 h after IR, but it was reduced following the DNA repair. This was consistent with the finding that apoptosis of germline cells was present in the seminiferous tubules after IR, especially at 1 h (IF and TUNEL). Apoptosis was also present in the PLZF(+) spermatogonia, particularly at 1 h after IR. Apoptotic cells decreased with the increase in DNA repair time after IR. Moreover, the caspase-3 protein was expressed in the undifferentiated spermatogonia following IR. The expression of caspase-3, P53, Ku70 and DNA-PKcs in the cultured spermatogonia was also up-regulated following IR in vitro, but their expression decreased gradually over time after IR, which was supported by the findings from flow cytometry, and the apoptosis of spermatogonia peaked at 24 h post IR. Conclusions: IR may induce the apoptosis of spermatogonia at early stage in vivo, but the apoptosis of spermatogonia secondary to IR occurs at a relatively later time point (24 h) in vitro mainly. The apoptosis of spermatogonia is improved over time after IR.