To explore the effect of N-acetyltransferase on the biological characteristics of Streptococcus agalacae (S. agalactiae), the nat gene deletion strain Δnat was constructed by homologous recombination in the background of HN016. The growth curve, morphology, cell adhesion, invasion, antiphagocytosis, whole blood survival and pathogenicity of wild type and Δnat strains were compared. The results showed that the hemolytic capacity of Δnat decreased and the loss of nat caused a decrease in the adhesion and invasion of S. agalactiae to tilapia brain cells and increased bacterial susceptibility to blood killing and phagocytosis. The result of challenge experiment in tilapia showed that Δnat (2×108 CFU/fish, the mortality rate is 36%) was significantly less lethal than HN016 (2×108 CFU/fish, the mortality rate is 85%). And the bacterial loads of Δnat showed 173-fold, 13.2-fold and 16-fold lower in blood, spleen and brain tissue, respectively, compared to that of HN016 at 12 hours after infection. This study firstly reports that NAT plays an important role in S. agalactiae resistance to host blood and phagocyte clearance, and assists in bacterial invasion and colonization of tilapia tissues.