Abstract: Spermatogenic cell heterogeneity is determined by the complex process of spermatogenesis differentiation. However, effectively revealing the regulatory mechanisms underlying mammalian spermatogenic cell development and differentiation via traditional methods is difficult. Advances in technology have led to the emergence of many single-cell transcriptome sequencing protocols, which have partially addressed these challenges. In this review, we detail the principles of 10x Genomics technology and summarize the methods for downstream analysis of single-cell transcriptome sequencing data. Furthermore, we explore the role of single-cell transcriptome sequencing in revealing the heterogeneity of testicular ecological niche cells, delineating the establishment and disruption of testicular immune homeostasis during human spermatogenesis, investigating abnormal spermatogenesis in humans, and, ultimately, elucidating the molecular evolution of mammalian spermatogenesis.