In order to investigate the dietary vitamin B6 (VB6) requirement for juvenile golden pompano, an experiment of six different diets with six dietary VB6 levels (0, 3.75, 7.47, 10.6, 13.7 and 18.5 mg/kg) was conducted. The results indicated that the content of dietary VB6 significantly increased weight gain rate (WGR), specific growth rate (SGR) and feed conversion ratio (FCR; p < 0.05). Golden pompano fed diet containing 7.47 mg/kg VB6 achieved the maximum ALT, AST, GPx, hepatic VB6 concentrations as well as the minimum MDA, however, the ALT, AST, GPx and hepatic VB6 concentrations decreased while MDA increased with a further increase in dieatry VB6. The highest values of GR and POD occurred at the 10.6 and 13.7 mg/kg dietary VB6 (p < 0.05) respectively. A diet supplemented with 7.47 mg/kg VB6 increased intestinal Na+, K+-ATPase, Chymotrypsin, γ-glutamyl transpeptidase, creatine kinase and amylase activities (p < 0.05). The relative abundance of Tenericutes and Bacteroidetes decreased while Proteobacteria and Firmicutes improved with an increase in dietary VB6 levels up to 7.47 mg/kg. Quadratic regression analysis on WGR, hepatic VB6 concentrations, AST and ALT indicated that the optimum dietary VB6 levels for juvenile golden pompano were 8.84–9.28 mg/kg.