Abstract: Clarias fuscus, a common freshwater fish in China, was selected as our experiment material. Two-day juvenile C. fuscus was divided into two groups. They were immersed in different doses of 17-methyltestosterone (50, 100, and 200 g/L 17-MT) or fed with 17-estradiol (100, 200, and 300 g/g 17-E2) for 30 days. Effects of 17-MT and 17-E2 on survival rate, sex ratio, gonad histology, and Foxl2 and Cyp19a1b expressions were examined by morphologic observation, histology and Real time fluorescent quantitative PCR during its period of sex differentiation (230d after hatching). The results showed that 17-MT and 17-E2 had no influence on survival rate but affected sex ratio and the time of gonadal differentiation. Doses of 17-MT at 100 and 200 g/L produced more males (70% and 76%, respectively) than 50 g/L 17-MT (54%) did (P0.05), but no significant difference in sex ratio was observed between the 50 g/L 17-MT treated group and the control group (56%). In addition, the former accelerated the occurrence of primary spermocytes for three and five days, but deferred that of ovarian cavity for four and six days, and primary oocytes for eight and nine days, respectively. In contrast, doses of 17-E2 at 200 and 300 g/g produced more males (74% and 78%, respectively) than the control group (P0.05), but no significant difference in sex ratio was observed between the 100 g/g 17-E2 treated group (66%) and the control group (56%). Dose of 17-E2 at 200 and 300 g/g accelerated the occurrence of ovarian cavity for two and three days, and primary oocytes for one and three days, respectively, but deferred that of primary spermatocytes for three and seven days. The expressions of Foxl2 and Cyp19a1b showed that dose of 200 g/L 17-MT increased the expression of Cyp19a1b but inhibited that of Foxl2.However, dose of 300g/g 17-E2 increased the expression both of Cyp19a1band Foxl2.The results suggested that Foxl2 but not Cyp19a1b involved in mediating sex differentiation directly in C. fuscus, and 17-MT inhibited but 17-E2 promoted the expressions of Foxl2 toinfluence the estrogen biosynthesis, which controlled the sex differentiation. However, Cyp19a1b played an indirect role on sex differentiation by acting on the hypothalamic-pituitary-gonadal axis.