Estrogen plays an important role during the regulation of ovarian development in crustaceans, and tamoxifen (TAM) is a commonly used estrogen antagonist for the studying of molecular mechanism between estrogen and ovarian development of crustacean. This study was designed to investigate the effect of estrogen antagonist on survival rate, gonadosomatic index (GSI), hepatosomatic index (HSI), and expressions of the related genes to ovarian development of female Portunus trituberculatus by in vivo experiment, culture experiment and quantitative PCR (qPCR). The results showed that different concentrations of TAM had no significant effects on the survival rate and HSI among all treatments, but could suppress GSI. The results of qPCR showed that TAM had significant effects on the expression levels of Vg (vitellogenin), ERR (estrogen related receptor), RXR (retinoid X receptor) and EcR (ecdysone receptor) genes in different tissues of P. trituberculatus. The expression levels of Vg gene in ovary and hepatopancreas were obviously inhibited by TAM. For ERR gene, low concentration of TAM (6.7 and 13.4 μg/g) could promote the ERR transcription in the ovary and thoracic ganglion, while different from the results of low concentration, the significant decrease was found on the ERR mRNA levels in the most examined tissues from the high concentration of TAM treatment (20 μg/g) compared to the control treatment. The expression profiles of EcR affected by TAM were similar to ERR in ovary and hepatopancreas. In addition, the low concentration of TAM could promote the expression of RXR gene in ovary, but no significant differences were found on RXR expression levels in the hepatopancreas. In conclusion, these results suggested that TAM could inhibit the Vg expression levels, possibly resulting in the low GSI of female P. trituberculatus, and ERR and EcR might be involved in regulatory processes of estrogen or exogenous estrogen antagonist on ovarian development.