<p>Spermatogenesis is a complicated process of sexual reproduction, involving cell proliferation and differentiation, and depends on intricate interactions between testicular somatic cells and germ cells. For further investigations on male germ cells’ development and differentiation in Chinese soft-shelled turtle (<i>Pelodiscus sinensis</i>), it is crucial to define testicular cell types and their molecular regulators. Here, the 10 × Genomics single-cell RNA sequencing was adopted to study the transcriptomic profiles of single cells during the spermatogenesis of adult Chinese soft-shelled turtle. In total, 7317 individual cell transcriptomes were collected for analysis, and 11 cell types were identified with known differentially expressed genes, including Leydig cells, Sertoli cells, spermatogonia, spermatocytes, and spermatids. Likewise, the top 10 marker genes and top-enriched gene pathways were analyzed in each cell type. Intriguingly, ligand-receptor analysis showed that the strongest interaction between Leydig cells and germ cells was using CellChat. Moreover, a primary developmental trajectory of male germ cells was constructed from spermatogonia to spermatids, as well as some important cell-specific regulators were identified for labeling the germ cells at different stages, including PCNA and Stra8, validated by immunostaining fluorescence. In addition, the transcriptomic profiles of male germ cells at different stages were comparatively analyzed among fruit fly, fish, mammals, and Chinese soft-shelled turtle, and the conserved and divergent regulators of male germ cells were summarized across species. In conclusion, this study provided novel insights into the testicular cells’ atlas in turtles, and the findings would facilitate the development of techniques for manipulating germ cells, such as isolating cells and defining stages of differentiation.</p>