Abstract: Genetic sex determination (GSD) along with environmental sex determination (ESD) are two crucial sex determination mechanisms. This study investigated gonadal differentiation mechanisms under 3 different photoperiod conditions (long light photoperiod: L﹕D=8﹕16; short light photoperiod: L﹕D=16﹕8; control: L﹕D=12﹕12) of black rockfish (Sebastes schlegelii) larvae, which are demersal at coast in the mid and high latitudes, at 35 dpb (day post birth). The result showed that the unnatural photoperiod conditions impacted the gonadal differentiation and development with the short light photoperiod condition to induce gonad masculinization. Estrogen level (E₂) first reached the peak value in the short light photoperiod condition, while androgen (T) level reached the peak value at 9d after treatment in all three conditions. The short light photoperiod condition significantly reduced the expression of the ovary differentiation related genes such as ERα, ERβ2 and foxl2 (P<0.05). Consider that the testis differentiation time was relatively late, the testis differentiation related genes of sox3, sox9, amh, and dmrt1 did not impact by photoperiod. In conclusion, short photoperiod could affect the gonad differentiation and development by restrain the ovary differentiation related genes expression and induce primitive gonad masculinization.