Objective To determine if granulocyte colony-stimulating factor (G-CSF) could prevent loss of spermatogenesis induced by busulfan chemotherapy via protection of undifferentiated spermatogonia, which might serve as an adjuvant approach to preserving male fertility among cancer patients. Design Laboratory animal study. Setting University. Animals Laboratory mice. Intervention(s) Five week-old mice were treated with a sterilizing busulfan dose and with 7 days of G-CSF or vehicle treatment and evaluated 10 weeks later (experiment 1) or 24 hours after treatment (experiment 2). Main Outcome Measure(s) Experiment 1 - testis weights, epididymal sperm counts, testis histology. Experiment 2 - PLZF immunofluorescent co-staining with apoptotic markers. Molecular analysis of G-CSF receptor expression in undifferentiated spermatogonia. Results Ten weeks after treatment, busulfan-treated mice that also received treatment with G-CSF exhibited significantly better recovery of spermatogenesis and epididymal sperm counts than animals receiving busulfan alone. G-CSF led to increased numbers of PLZF+ spermatogonia 24 hours after treatment that was not accompanied by changes in apoptosis. To address the cellular target of G-CSF, mRNA for the G-CSF receptor, Csf3r, was found in adult mouse testes and cultured Thy1+ (undifferentiated) spermatogonia, and cell-surface localized CSF3R was observed on 3% of cultured Thy1+ spermatogonia. Conclusion(s) These results demonstrate that G-CSF protects spermatogenesis from gonadotoxic insult (busulfan) in rodents and this may occur via direct action on CSF3R+undifferentiated spermatogonia. G-CSF treatment might be an effective adjuvant therapy to preserve male fertility in cancer patients receiving sterilizing treatments.