Abstract: Investigating the best acclimation temperature conditions for Epinephelus fuscoguttatus ♀×E. lanceolatus ♂ can provide a theoretical basis for its survival and circulation. We decreased the water temperature to 24, 20, 18 and 16 ℃ at the rate of 0.5−1.0 ℃·h⁻¹, then recorded the physiological responses and survival time, as well as the changes of water quality, non-specific immune index and antioxidant indicator. The results show that the survival time of the grouper increased with the decrease of temperature within the range of 24−16 ℃. When the water temperature was maintained at 16 ℃, the concentration of total ammonia nitrogen (TAN) increased slowly, and the survival time was the longest, up to 109 h. The non-specific immune and antioxidant parameters changed after the grouper being cultured at different temperatures for 48 h. Compared with the control group, the contents of complement protein 3 (C3), globulin (GLB) and total protein (TP) increased in 16 ℃ group significantly, while the other indexes had no significant changes. The activities of superoxide dismutase (SOD) and catalase (CAT) in the liver tissue in 16 ℃ group increased significantly. However, the activity of glutathione peroxidase (GPx) decreased significantly, but higher than those of the other temperature groups. Compared with the other temperature treated groups, the content of malondialdehyde (MDA) decreased significantly. Thus, 16 ℃ temporary culture can not only maintain good water quality, but also reduce the metabolic level of fish and maintain the homeostasis of internal environment, so as to reduce the damage caused by oxidative stress reaction. It is suitable for temporary culture and transportation of Epinephelus fuscoguttatus ♀×E. lanceolatus ♂.