Grouper Epinephelus malabaricus (weighing 46.37 ± 5.10 g) previously maintained in 24‰ seawater were transferred to 14, 19, 24 (control), 29, and 34‰ seawater. Serum cortisol, glucose, lactate, and osmolality levels were measured at 7 time points during 240 min. Serum cortisol and glucose levels of fish transferred to 29 and 34‰ seawater significantly increased to the highest after 10 and 20 min, respectively. No significant differences in serum cortisol and glucose levels were observed for the fish after 30 min among all treatments. Serum lactate level of fish transferred to 14, 19, 29, and 34‰ seawater was significantly lower than that of the control fish after 10-30 min. However, no significant differences in serum lactate were observed 60 min among five treatments. The serum osmolality of the fish following 240-min transfer increased directly with salinity, whereas the osmoregulatory capacity value (medium osmolality-plasma osmolality) of the fish following 240-min transfer was inversely related to salinity. It is concluded that grouper showed strong osmoregulation in 14-34‰ seawater. Serum cortisol and glucose levels of fish transferred to 29 and 34‰ seawater increased rapidly in 10-30 min, indicating an early stress response.