A trial was conducted to determine the effects of dietary Vitamin C levels on hypothalamic-pituitary-interrenal axis and physiological indices of juvenile hybrid grouper (Epinephelus lanceolatu♂×Epinephelus fuscoguttatus♀) under acute low temperature stress. A basal diet was used as a control, and four other diets were prepared by supplementing 38, 76, 152, or 304 mg/kg vitamin C. Triplicate groups of fish (initial body weight, 50 g) were fed one of test diets at ratio of 1.5% or 2% body weight for 12 weeks at 27-28℃. Uniform sizes of fish were selected for acute low temperature stress (17℃) at the end of rearing management. Some biochemical indices in their serum and corticotrophin-releasing factor content of brain were examined over 2 hours. Results showed that:Weight gain of the fish fed with D38 group was significantly higher than that of D152 and D304 groups. We found no significant difference in corticotropin releasing factor content (CRF), adrenocorticotropic hormone content (ACTH), epinephrine content (EPI) or cortisol content in juvenile hybrid grouper, among all treatment groups. After acute low temperature stress, CRF content was increased by increasing dietary vitamin C levels. While ACTH and EPI content in serum showed a reverse trend, fish fed diets with vitamin C 304 mg/kg was significantly lower than control diet. Cortisol was reduced significantly in fish fed diets 76 mg/kg to 304 mg/kg diets compared to control diet. Fish fed diets 152 mg/kg to 304 mg/kg dites T3 content were significantly higher than control diet. Whereas, after acute low temperature stress, fish fed diets 38 mg/kg to 304 mg/kg were significantly higher than control diet. There was no significant difference in T4 content in fish among different treatments. Fish fed diet 304 mg/kg diet showed significantly higher than other groups, whereas no difference was found among group D0 to D152. TSH content in D76 group exhibited obviously higher than D0, D38 or D304 groups. After acute low temperature stress, all diets containing vitamin C significantly elevated TSH content in serum. Dietary vitamin C levels had no strong effects on GCK and HK contents of liver. After acute low temperature stress, GCK content was negatively correlated with dietay vitamin C, and reached lowest value in 304 mg/kg diet, and was significantly lower than other groups. HK content reached lowest value in 76 mg/kg diet, and was significantly lower than D0, D152, D304 groups. Significantly higher PEPCK value was found in fish fed 304 mg/kg vitamin C compared to 0 mg/kg diet. After acute low temperature stress, PEPCK concentration was decreased significantly with increasing dietary vitamin C levels. All those data reveal new insights into the funtion of vitamin C that VC can modulate some biochemical indices of hypothalamic-pituitary-interrenal axis, protect and regulate the thyroid of fish, and maintain the dynamic balance of fish body under acute low temperature stress.