Background <p>Sex reversal is an intricate and fascinating process orchestrated by a finely organized network. The orange-spotted grouper (<i>Epinephelus coioides</i>) is a protogynous hermaphroditic fish, but the regulatory mechanism of its sex reversal is not yet well understood. </p> Results <p>Here, single-cell transcriptome sequencing (scRNA-seq) of the gonads was performed during sex reversal induced by 17α-methyltestosterone (MT). Based on the scRNA-seq data, various gonadal cell types and subtypes, including eight types of germ cells and nine types of somatic cells, were characterized and identified. Moreover, a single-cell atlas of dynamic changes in gene expression during sex reversal in orange-spotted grouper was constructed. We outlined the precise trajectories of germ stem cells (GSCs) and male somatic cells, which revealed that new male germ cells were originated from a subcluster of GSCs with increased levels of spermatogenesis genes. Moreover, we revealed that <i>piwil1</i> and <i>dmrt1</i> were important for the male-oriented differentiation of GSCs. Additionally, oocytes exhibited abnormal and significant apoptosis and active proliferation during the early stage of sex reversal.</p> Conclusions <p>Overall, these results address the questions of how male germ cells are generated and how female germ cells disappear during sex reversal in orange-spotted grouper. These findings profile a comprehensive dynamic map of gonadal cells during sex reversal, which can provide new insights into sex change in lower vertebrates.</p>