Six practical diets were formulated to contain 2.6 (the basal diet), 48.9, 95.5, 189.83, 382.4, 779.53 mg/kg vitamin C diet. After 8 weeks of feeding, growth performance, antioxidant status, and innate immunity of juvenile pufferfish were analyzed. Results showed that specific growth rate and protein efficiency ratio in fish fed diets supplemented with vitamin C were significantly higher than in fish fed the control diet. Dietary vitamin C significantly up-regulated the expression levels of growth hormone receptors and Insulin-like growth factor. Blood cell numbers and respiratory burst activity were enhanced in fish fed vitamin C diet. Serum alkaline phosphatase, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase activity were significantly influenced by the dietary vitamin C levels. Fish fed diets supplemented with vitamin C showed increased hepatic superoxide dismutase, glutathione peroxidase, and catalase activity, but decreased malondialdehyde activity. In addition, we found that dietary vitamin C can significantly up-regulate the expression levels of immune-related genes (HSP70, HSP90, BAFF, TNF-α and IL-6) in liver. These results indicate that dietary vitamin C can significantly influence growth performance, antioxidant status, and innate immunity of pufferfish.