To determine the optimum water temperature (a temperature which minimizes metabolic activities and stress in fish) for long distance transportation of red-spotted grouper (Epinephelus akaara), by evaluating the behavior, survival and physiological and hematological responses to variable water temperature conditions (9, 12, 15, 18 and 21℃) for 48 hours. Fish exposed to 9℃ died at 48 hours exposure, but those exposed to 12, 15, 18 and 21℃ treatment groups all survived. Fish in the 15, 18 and 21℃ exposure groups exhibited normal swimming, while those exposed to 12℃ were observed to be stationary at the bottom of the tank. The plasma cortisol and glucose concentration were higher in fish exposed to 12℃ than at other temperature conditions. The fish in the 12 and 15℃ groups had significantly higher hematocrit (Ht) and hemoglobin (Hb) than those in the 18 and 21℃ groups (P<0.05). Levels of aspartate aminotransferase (AST), NH3 and osmolality showed a pattern similar to the levels of plasma cortisol, while alanine aminotransferase (ALT) and total protein did not significantly vary among the experimental groups. These results indicate that the optimum water temperature for long distance transportation of the red-spotted grouper is 15℃.