The aim of this paper is to investigate the impact of high temperature stress on serum biochemical parameters and histopathology of Nile tilapia Oreochromis niloticus infected by Streptococcus agalactiae. The average weight of Nile tilapia was (62.0±3.33) g. They were cultured on two temperature levels: 29 ℃ (the optimal water temperature for the growth of tilapia) group and 33 ℃ (high temperature stress) group. All fish were acclimated for one week in the laboratory then artificially infected by S. agalactiae. The cumulative mortality was recorded at different time post infection; Blood and tissue samples were respectively collected at 0, 12, 24, 48, 96 and 120 h after infection. Then they were examined. The result showed that cumulative mortality in 33 ℃ group was significantly higher than that of 29 ℃ group; Glutamic-pyruvic transaminase(ALT) activity of fish in 33 ℃ group was significantly higher than those fish in 29 ℃ group at 96 h after infection, while glutamic-oxaloacetic transamnase(AST) activity of fish in 33 ℃ group was significantly higher than those fish in 29 ℃ group at 48 h after infection; Potassium-ion (K⁺) and sodion (Na⁺) of fish at 120 h after infection were significantly higher and lower respectively than those fish prior to infection in 33 ℃ group, but that were not significantly different compared with those fish prior to infection in 29 ℃ group. Creatinine (CREA) activity of fish in 33 ℃ group was significantly higher than those fish in 29 ℃ group at 12 h after infection. Albumin/globulin(A/G)index of fish in 33 ℃ group was significantly lower than those fish in 29 ℃ group at 120 h after infection. Alkaline phosphatase (AKP) activity of fish was increased with the progress of infection in 29 ℃ group, while it was increased and then decreased in 33 ℃ group, but the peak time in 33 ℃ group was earlier than that in 29 ℃ group. Superoxide dismutase (SOD) activity was increased first and then decreased, finally it was increased once again. Histopathology showed that hepatic cord arranged irregularly, while the spleen and renal glomerular hemorrhaged slightly under high temperature stress; The spleen was severely congested at 12 h after infection and they were remarkably broken and obviously amyloidosis at 24 h after infection in both groups. Renal glomerular atrophy, degeneration and necrosis in renal tubule epithelial cell were obvious at 48 h after infection in both groups. Macrovesicular steatosis and hyaline degeneration in hepatocyte were commonly observed at 96 h after infection in 33 ℃ group, but the same histopathological changes in liver and kidney were only found at 120 h after infection in 29 ℃ group. These results indicated that increased susceptibility of Nile tilapia to S. agalactiae resulted from the immunosuppression caused by high temperature stress. Spleen might be the first target organ specified to this pathogen. The injury of liver was accelerated by high temperature stress and liver damage was more serious in the late infection.