Abstract: Streptococcosis, caused by Streptococcus agalactiae, seriously harms the Chinese tilapia aquaculture industry. Improving the internal disease resistance of tilapia is a preferred method to control this disease. We observed the physiological responses of hybrid tilapia (Oreochromis niloticus♀×O. aureus♂) and their parents after challenged with pathogenic S. agalactiae. The cumulative mortality was recorded at different time points. Blood and spleen samples were collected at 0, 7h, 24h, 48h, 72h, 120h and 168h after infection, respectively. Blood physiological and biochemical indexes were measured between hybrid tilapia and its parents. The expression of pro-inflammatory cytokines in spleen were compared to gain an in-depth understanding of the physiological and molecular basis of the disease resistance heterosis. The result showed that the cumulative mortality of hybrid tilapia was significantly lower than its parents. The white blood cell count, red blood cell count and hematocrit of hybrid tilapia were the highest of three tilapias before infection. The white blood cell count increased significantly after infection, whereas hematocrit and hemoglobin decreased significantly. The infection of fish resulted in a strong inhibition of the respiratory burst, and the blood physiological indexes of hybrid tilapia were closer to those of O. aureus. The result showed that the cumulative mortality of hybrid tilapia was significantly lower than their parents. The white blood cell count, red blood cell count and hematocrit of hybrid tilapia were the highest of three tilapias before infection. The white blood cell count increased significantly after infection, whereas hematocrit and hemoglobin decreased significantly. The infection resulted in a strong inhibition of the respiratory burst, and the blood physiological indexes of hybrid tilapia were closer to those of O. aureus. Hybrid tilapia had higher glucose, lysozyme activity and the respiratory burst compared with their parents. The pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) mRNA levels in the spleen were significantly up-regulated after infection (P<0.05), which peaked at 7h, 48h and 7h, respectively. The TNF-α and IL-6 mRNA levels of hybrid tilapia was the highest of three tilapias before and after infection, and the IL-1β mRNA levels of hybrid tilapia was significantly higher than their parents before infection and at 7−48h after infection. The results showed that hybrid tilapia had stronger defense ability against S. agalactiae infection than their parents with significantly increased pro-inflammatory cytokines after infection. Taken together, this study indicated that the pro-inflammatory cytokines might be involved in host defense against bacterial infection, and the immune response of hybrid tilapia is more active than their parents to support disease resistance.