The rice field eel (Monopterus albus) is a protogynous hermaphroditic fish reared commercially as an aquaculture species in East Asia. Artificial breeding of rice field eels requires an abundance of adult females and males; however, the eels need to undergo a long period of female and intersexual stages before they develop into males, which results in a shortage of functional males in production practice. This study developed an artificial method for producing functional males through the oral administration of an aromatase inhibitor (AI, letrozole: 300 mg/kg feed) in juvenile fish. Histological analysis of gonadal sections of treated fish revealed the transition of primary ovaries to mature testes. During the initial period of the AI treatment, 2-month-old rice field eels were exclusively female. AI administration resulted in a significant reduction in 17β-estradiol levels and increase in testosterone and 11-ketotestosterone levels in vivo. Cell proliferation occurred in the outer layer of the gonadal epithelium, and oocytes degenerated in the ovaries. After 120 days of AI treatment, the ovaries were completely converted to testes. The sexually reversed fish maintained the male sex and produced functional sperm after cessation of drug treatment. Notably, artificially sex-reversed males' fecundity did not differ significantly from wild-type males. Quantitative PCR analysis revealed that the expression of cyp19a1a decreased, whereas the mRNA levels of estrogen receptors esr1, esr2a, and esr2b increased significantly during AI treatment. Moreover, the expression of foxl2 was downregulated, whereas dmrt1, amh, and sox9 were upregulated in the gonads of fish administered AI. This study revealed that endogenous estrogen synthesis plays a key role in the sex change of rice field eels, with ovaries undergoing a reversal to testes in response to estrogen insufficiency. Such artificially induced sex conversion will be of considerable value in breeding rice field eels.