Background <p>The <i>Monopterus albus</i> serves as a unique model for studying sex reversal, transitioning naturally from female to male. However, the origins of male germline stem cells (GSCs) and the roles of somatic cells during sex reversal remain poorly understood.</p> Methods <p>We performed single-cell RNA sequencing (scRNA-seq) on ovarian, ovotestis, and testicular tissues to construct a gonadal cell atlas. RNA fluorescence in situ hybridization (RNA-FISH) validated cell subpopulations.</p> Results <p>Cell types of germ cells and somatic cells in gonads were identified, and their differentiation trajectories during sex reversal were depicted. Our results show that GSCs^{<i>wdr17+tep1−</i>} in the ovaries possess bipotential differentiation capacity and can transform into GSCs^{<i>wdr17+ tep1+</i>} with the capability to differentiating into sperm during sex reversal. Two theca cell subpopulations cooperate to synthesize steroid precursors in ovaries, with Leydig cells in testes likely originating from theca cell transitions. Proliferative <i>gdf9</i>^<i>+</i> follicle cells promoted folliculogenesis and persisted in male gonads with reduced <i>hsd17b1</i> expression. Stromal heterogen-eity analysis revealed <i>bmp4</i>^<i>+</i> mesenchymal stem cells (MSCs^<i>bmp4+</i>) as potential precursors for Sertoli cells in testes.</p> Conclusions <p>This study provides a comprehensive cellular roadmap of gonadal cell dynamics during sex reversal in <i>Monopterus albus</i>. Our findings unveil the molecular mechanisms underlying germline and somatic cell fate determination, offering novel insights into vertebrate sex reversal and potential therapeutic strategies for disorders of sex development (DSD).</p>