Abstract: The failure of ovaries to reach sexual maturity in XY genetic pseudofemale largemouth bass (Micropterus salmoides) represents a key technical bottleneck restricting the production of all-male production. To elucidate the mechanisms underlying gonadal developmental arrest and explore intervention strategies, we fed fish during the critical period of sex differentiation (20–75 days post hatching, dph) with diets supplemented with different concentrations of 17β-estradiol (E₂) and trilostane (Tri) to induce feminization. Histological results at 135 dph showed successful feminization across all treatments, with the 100 mg/kg group exhibiting the highest efficacy. Therefore, XY pseudofemales from this treatment group were selected for continued E₂ and Tri administration prior to the overwintering period (195–270 dph, October–January) and before the breeding season (330–370 dph, March–April), aiming to promote ovarian maturation. The results showed that at 340 dph, the plasma 11-ketotestosterone (11-KT) concentrations in the 25 mg/kg (ET25) and 50 mg/kg (ET50) XY pseudofemale treatment groups were lower than that in the control XY pseudofemales (CXY). At 370 dph, the E₂ and vitellogenin concentrations in ET25 and ET50 were higher than those in CXY, while their mesenteric fat coefficients were reduced. Furthermore, compared with XX genetic females, the expression of genes involved in the endogenous steroid synthesis pathway (star, cyp11a1, cyp17a1, cyp19a1a), key receptors (esr1, fshr), and neutral lipid droplet accumulation (lpl) was suppressed in both the treatment groups and the control XY pseudofemales, which may be a key reason why their ovarian development remained arrested at the cortical alveolus stage (stage II). Re-administration of E₂ and Tri from pre-overwintering to pre-breeding periods effectively suppressed the elevation of plasma 11-KT levels, maintained relatively high E₂ levels, and simultaneously promoted the utilization of mesenteric fat and vitellogenin synthesis. Future investigations should focus on receptors and upstream regulatory genes, hormone signals associated with the ovarian transition from stage II to stage III, as well as the mechanisms of nutrient allocation and utilization in vivo, to provide a theoretical basis for promoting normal ovarian development in XY genetic pseudofemale largemouth bass.