The effects of osmotic stress on the non-specific immune response of Nile tilapia, Oreochromis niloticus, were investigated. Osmoregulatory mechanism of tilapia has been studied, but less information is available about innate immune response of O. niloticus faced with hyperosmolality. Acute osmotic stress was elicited by transferring tilapia from freshwater (FW) to 24 psu seawater (SW). Non-specific immune parameters including lysozyme activities of plasma and head kidney (HK), alternative complement pathway (ACP) activity in plasma, phagocytic capacities of spleen and HK immune cells, and respiratory burst activity of immune cells in both HK and spleen were analyzed. Lysozyme activities were increased at 1 h and 30 h after transfer to SW, but decreased at 10 h after SW transfer. Conversely, ACP activity increased 10 h after SW transfer. Phagocytic capacity increased slightly at 1 h and 5 h after SW transfer, and respiratory burst activity showed an increase in superoxide release at 10 h after SW transfer. Taken together, these results indicate that the exposure of tilapia to hyperosmotic conditions has immunostimulatory effects on cellular and humoral immune reactions.