The sexual size dimorphism of the Chinese tongue sole (Cynoglossus semilaevis) has greatly obstructed its sustainable development; however, the underlying mechanism remains unclear. Based on C. semilaevis transcriptomic information, 24-dehydrocholesterol reductase (dhcr24) was identified in steroid biosynthesis, showing female-liver-biased expression. Dhcr24 has been reported to participate in various processes, such as cholesterol synthesis, oxidative stress response, neuroprotection, and cell survival. The present study assessed its role in the sexual size dimorphism in fish. First, detailed expression pattern analysis showed that dhcr24 mRNAs were extensively expressed in tissues and the highest levels were found in the liver and gonads of females. Analysis of the dhcr24 promoter region demonstrated different DNA methylation statuses in female, male, and pseudomale gonads with higher epigenetic modification in males. The confirmation of transcription activity of the dhcr24 promoter and putative transcription factors (e.g., ER, AR, SREBP, and POU1F1a) provides the foundation for studying its regulatory mechanism. Finally, dhcr24-siRNA mediated knock-down assay using C. semilaevis liver cells showed that steroid biosynthesis related genes (e.g., ebp, dhcr7, and sc5d), core component of PI3K/Akt pathway (e.g., pi3k), and igf1r exhibited different expression patterns. Further investigation on the interplay between steroid hormones, dhcr24, PI3K/Akt, and IGF-1 systems will be valuable to better understand the mechanism underlying the sexual size dimorphism in C. semilaevis.