Spermatogenesis is a complicated course of several rigorous restrained steps that spermatogonial stem cells undergo to develop into highly specialized spermatozoa; however, specific genes and signal pathways, which regulate the amplification, differentiation and maturation of these cells, remain unclear. We performed bioinformatics analyses to investigate the dynamic changes of the gene expression patterns at three time points in the course of the first wave of rat spermatogenesis. Differently expressed genes (DEGs) were identified, and the features of DEGs were further analyzed with GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes) and Short Time-series Expression Miner (STEM). A total of 2954 differentially expressed genes were identified. By using STEM, the top 10 key genes were selected in the profile according to the enrichment results, and the distinguishable biological functions encoded by these DEGs were automatically divided into three parts. Genes from 6, 8 and 10 days were related to biosynthesis, immune response and cell junction, and genes from 14, 15 and 16 days were related to energy metabolic pathways. The results also suggest that genes from 29, 31 and 35 days may shift metabolic to sperm motility, sperm flagellum and cilium movement.