Objective To explore the dynamic responses of Sertoli cells to depletion of spermatogonial stem cells by busulfan. Methods After intraperitoneal injection of 15, 30 or 44 mg/kg busulfan to mice, the spermatogenesis and the expression of GDNF, PLZF, Nanog and GFRɑ1 mRNA were assessed by real-time quantitative PCR at 5 and 28 days after the busulfan treatment. Results Glial cell line-derived neutrophic factor(GDNF) was significantly increased and showed a dose-dependent trend at 5 days after busulfan treatment, but no significant difference was seen in the expression of promyelocytic leukemia zinc finger(PLZF) and GDNF family receptor α-1(GFRα1). The testicular histology also appeared no significant difference at 5 days after busulfan treatment. At 28 days after busulfan treatment, the relative expression levels of GDNF, PLZF, Nanog and GFRɑ1 mRNA were drastically increased. Morphological observation showed that spermatogenesis damages became even more severe as the busulfan dose increased. Conclusions Sertoli cell response to the depletion of spermatogonia occurs as early as the fifth day after busulfan treatment. Production of GDNF in Sertoli cells shows a compensatory increase, which may stimulate spermatogonial stem cells to accelerate their self-renewal, reflected by the enhancing expression of Nanog and PLZF, and ultimately promote the restoration of spermatogenesis.