Long-chain polyunsaturated fatty acids (LC-PUFA) play key roles in sex steroid hormone synthesis in fish. Regarding docosahexaenoic acid (DHA), currently the regulating effects were mostly evaluated in female fish rather than males. This study aimed to investigate the DHA effects on the sex steroid hormone production in male Chinese tongue sole. Three experimental diets were prepared, containing different DHA levels: a control (C) group (5.16% DHA of total fatty acids [TFA]), a low DHA group (DHA-L, 8.93%), and a high DHA group (DHA-H, 16.47%). A 58-day feeding experiment was conducted, and each diet was fed to triplicate tanks of fish. Additionally, an in vitro study with Leydig's cells of this species was conducted to validate the in vivo results. The concentration of testosterone (T) and 11-ketotestosterone (11-KT) in the serum increased with increasing levels of dietary DHA. Dietary DHA significantly upregulated the expression of steroid hormone biosynthetic genes