Gossypol is known to be a polyphenolic compound toxic to animals. However, its molecular targets are far from fully characterized. To evaluate the physiological and molecular effects of gossypol, we chose turbot (Scophthalmus maximus L.), a carnivorous fish as our model species. Juvenile turbots (7.83 ± 0.02 g) were fed with diets containing gradient levels of gossypol at 0 (G0), 600 (G1) and 1,200 (G2) mg/kg diets for 11 weeks. After the feeding trial, fish growth, body protein and fat contents were significantly reduced in G2 group compared with those of G0 group (P< 0.05). Gossypol impacted little on digestive enzyme activities and intestine morphology. However, gossypol caused liver fibrosis and stimulated chemokine and pro-inflammatory cytokine secretions. More importantly, gossypol suppressed target of rapamycin (TOR) signaling and induced endoplasmic reticulum (ER) stress pathway in both feeding experiment and cell cultures. Our results demonstrated that gossypol inhibited TOR signaling and elevated ER stress pathways both in vivo and in vitro, thus providing new mechanism of action of gossypol in nutritional physiology.