To clarify the functions and mechanism of stimulated by retinoic acid gene 8 (Stra8) in spermatogenesis, we analyzed the testes from Stra8 knockout and wild-type mice during the first wave of spermatogenesis. Comparisons showed no significant differences in morphology and number of germ cells at 11 days postpartum, while 21 differentially expressed genes (DEGs) associated with spermatogenesis were identified. We speculate that Stra8 performs many functions in different phases of spermatogenesis, such as establishment of spermatogonial stem cells, spermatogonial proliferation and self-renewal, spermatogonial differentiation and meiosis, through direct or indirect regulation of these DEGs. We therefore established a preliminary regulatory network of Stra8 during spermatogenesis. These results will provide a theoretical basis for further research on the mechanism underlying the role of Stra8 in spermatogenesis.