Objective To establish a mouse model treated with busulfan and to investigate its effects on the metabo-lism of spermatogonial stem cells(SSCs)of mouse testis.Methods C57BL/6J male mice with age of 8 weeks were injected with 10 mg/kg of busulfan intraperitoneally,then Thy1 positive cells were selected by immunomagnetic beads on day 0,day 5 and day 10 and followed by identification for purity and metabolomic analysis.Results The testis weight ratio decreased and the tissue structure of testis was damaged(P＜0.05).Based on the results of principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA),there were signifi-cant metabolic differences between the sample groups treated for 0 d,5 d and 10 d.A total of 89 differential metabolites were identified including glutathione(GSH),arginine and unsaturatedfatty acids(UFAs),and their important metabolic pathways involved glycerophospholipid metabolism,arginine and proline metabolism.Conclu-sions Affecting the specific metabolic pathway may result in obvious reproductive toxicity and lead to decrease of testicular weight as well as tissue structure damage in mice.Metabolomic analysis showed that the potential repro-ductive toxicity mechanism of SSCs may be related to the metabolic pathways such as lipid metabolism,arginine and proline metabolism.