Infertility affects 10-15% of couples worldwide, and male factors account for 50%. Spermatogenesis is precisely regulated by genetic factors, and the mutations of genes result in abnormal spermatogenesis and eventual male infertility. The aim of this study was to explore the role and transcriptional regulation of P63 in the apoptosis and mouse spermatogenesis. P63 protein was decreased in male germ cells of P63(+/-) mice compared with wild-type mice. There was no obvious difference in testis weight, sperm motility, and fecundity between P63(+/-) and wild-type mice. However, abnormal germ cells were frequently observed in P63(+/-) mice at 2 months old. Notably, apoptotic male germ cells and the percentage of abnormal sperm were significantly enhanced in P63(+/-) mice compared to wild-type mice. Spermatogonia, pachytene spermatocytes and round spermatids were isolated from P63(+/-) and wild-type mice using STA-PUT velocity sedimentation, and they were identified phenotypically with high purities. RNA sequencing demonstrated distinct transcription profiles in spermatogonia, pachytene spermatocytes, and round spermatids between P63(+/-) mice and wild-type mice. In total, there were 645 differentially expressed genes (DEGs) in spermatogonia, 106 DEGs in pachytene spermatocytes, and 1152 in round spermatids between P63(+/-) mice and wild-type mice. Real time PCR verified a number of DEGs identified by RNA sequencing. Gene ontology annotation and pathway analyzes further indicated that certain key genes, e.g., Ccnd2, Tgfa, Hes5, Insl3, Kit, Lef1, and Jun were involved in apoptosis, while Dazl, Kit, Pld6, Cdkn2d, Stra8, and Ubr2 were associated with regulating spermatogenesis. Collectively, these results implicate that P63 mediates the apoptosis of male germ cells and regulates three stages of spermatogenesis transcriptionally. This study could provide novel targets for the diagnosis and treatment of male infertility.