Nile tilapia is a GSD + TE (genotypic sex determination plus temperature effect) fish species. However, the role of 17β-estradiol (E2) during high-temperature-induced masculinization is unknown in fish species exhibiting GSD + TE. Because letrozole can specifically downregulate E2, the differences of the E2 level and sex ratio between the high-temperature-treated groups and various doses of letrozole treated groups could help quantitatively analyze the role of E2 in Nile tilapia GSD + TE. The results indicated that high-temperature could also significantly downregulate E2, and the level of E2 in the XX + HT (9 days postfertilization (dpf) larvae cultured in 36 °C water for 12 days) group at 21 dpf was only approximately half that in the XX group. Comparison of E2 and the sex reversal ratio between the high-temperature treated group and various letrozole treated groups showed that the level of E2 in the XX + HT group at 21 dpf was almost the same as that in the XX + L1.5 (9 dpf XX larvae cultured in 28 °C water with1.5 μg/L letrozole for 12 days) group, but the 70.00% male ratio in the XX + HT group was significantly higher than that in the XX + L1.5 (58.33%) group and was similar to that in the XX + L4.5 groups (66.25%) and the XX + L13.5 group (77.08%). Comparison of E2 and the female ratio between the high-temperature treated group and the HT + E2 compensation groups further indicated that the E2 level in the XX + HT + E:4 (9 dpf fry cultured in 36 °C water containing 4 μg/L E2 for 12 days) group was similar to that in the XX group at 21 dpf, and the female ratio was close between the two groups. qRT-PCR and western blot analysis showed that the high-temperature treatment significantly downregulated the expression of ovary-type P450 aromatase (cyp19a1a) and upregulated the expression of the male sex differentiation genes doublesex and mab-3 related transcription factor 1 (Dmrt1) at 21 dpf. Additionally, the high-temperature treatment also down-regulated Nile tilapia gonad aromatase activity. Collectively, the present study uncovered the role of E2 during high-temperature–induced Nile tilapia female sex reversal into pseudomales (phenotypic males) and the regulatory effect of high-temperature on the E2 pathway, providing an important foundation for understanding the mechanisms of high-temperature-induced Nile tilapia female sex reversal.