This study investigated the physiological effects of a diet containing calcium oxide (CaO) on the olive flounder, Paralichthys olivaceus. The results indicate that the amount of calcium in the blood was higher in the calcium group compared with the controls. The aspartate amino transferase (AST) and alanine amino transferase (ALT) levels were lower in the calcium group, while lysozyme activity was higher in the calcium group. Histologically, fish in the calcium group had more hepatocytes in the liver and more intact scales. In addition, an attack experiment using Vibrio anguillarum (KCTC-2711) and Edwardsiella tarda (KCTC-3657) showed that the survival was higher in the calcium group compared with the controls. These results suggest that feeding fish CaO when they require it, such as during periods of gonadal maturation and rapid growth, will increase their immunity and resistance to pathogens.