The busulfan-treated mouse model showed abnormal testis morphology and reduced sperm number and testis weight. Testicular and sperm damage was most severe at 30 days after busulfan treatment. The protein level of MGAT1 was increased in busulfan-treated mouse testis. The busulfan-treated mouse testes were also subjected to label-free quantification proteomics, which revealed 190 significantly downregulated proteins. Clustering heatmap, gene ontology, KEGG pathway and protein interaction analyses were performed and then validated by molecular experiments. An increased understanding of reproductive proteins function in vitro and in vivo will help to prevent and treat reproductive diseases.