Thiamethoxam has emerged as an environmental contaminant detected in aqueous environments, and its endocrine-disrupting effect at chronic exposure in teleosts remains unknown. In the present study, a docking experiment and an in vivo test were integrated to systematically explore the toxic mechanisms of thiamethoxam in fish. Histological analysis, plasma VTG and hormone level (E2, 11-KT, T3 and T4) determinations, and HPG and HPT gene expression quantification were performed after Chinese rare minnow (Gobiocypris rarus) was exposed to thiamethoxam (0, 0.5, 5, and 50 μg/L) for 90 days. According to the docking study, thiamethoxam had different interactions with ERα, AR and TRα via hydrogen bonding. A decrease in body length and plasma T4 was observed in both genders. The histological damage in liver and delayed gonadal development were observed in both genders at 50 μg/L thiamethoxam treatment. In males, the following HPG axis genes were upregulated: gnrh and cyp19b in the brain; vtg and cyp19a in the liver; and cyp17 and cyp19a in the gonad. In females, erɑ in the liver was significantly upregulated with 0.5 μg/L thiamethoxam treatment, and cyp17 in the gonad was upregulated with all treatment. The suppression of cyp19a, gnrh, cyp11a, and ttr was observed at the concentration of 5 μg/L in the female liver. Taken together, the endocrine system of Chinese rare minnow might be disrupted after chronic exposure to thiamethoxam.