In order to breed the all-female largemouth bass (Micropterus salmoides) population, 15-day old largemouth bass, with an average length of (15.1±0.09) mm, were fed dietary supplements containing 0 (MT0), 50 (MT50), and 100 mg/kg (MT100) 17α-methyltestosterone (MT). After continuous feeding for 60 days, the effects of MT on the growth, sex differentiation, hormone levels, and gene expression variations in largemouth bass were analyzed. The body lengths and weights of largemouth bass in the MT50 and MT100 groups were significantly lower than those in the MT0 group (P＜0.05). The male sex ratio in both MT50 and MT100 groups was 100%, which was significantly higher than that in the MT0 group (45%) (P＜0.05). Histological sections of the gonads indicated that the gonadal structures of MT-induced physiological males were similar to those of wild-type males. The concentration of estradiol in the physiological males of the MT50 and MT100 groups was significantly lower than that in the wild-type females of MT0 group (P＜0.05), while the concentration of testosterone in physiological males of the MT50 group was significantly higher than that in the females of MT100 and MT0 groups (P＜0.05). Additionally, compared to the wild-type females, the expressions of Dmrt1 and Gsdf were upregulated, while the expressions of Foxl2 and Cyp19a1a were downregulated in the gonads of physiological males in the MT50 and MT100 groups. In conclusion, dietary supplementations of 50~100 mg/kg MT could effectively induce the 15-day-old female largemouth bass sex-reversal into physiological males. This study establishes a physiological male induction method and provides valuable information for the breeding of all-female largemouth bass population.