This study was investigated to establish the dietary vitamin B12 requirement of sub-adult Pacific white shrimp. Six semi-purified diets were formulated by including cyanocobalamin at 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 mg/kg levels (designated as Con, Cb0.5, Cb1.0, Cb1.5, Cb2.0 and Cb2.5, respectively). The actual cyanocobalamin levels in the diets were 0.03, 0.48, 1.21, 1.78, 2.10 and 2.59 mg/kg, respectively. Total 216 shrimp (Initial weight: 12.94 ± 0.05 g) were randomly distributed to 18 tanks (300 L) in triplicate groups and fed the experimental diets for six weeks. Final mean body weight, weight gain (WG) percentage and specific growth rate were significantly increased in Cb1.0, Cb1.5, Cb2.0 and Cb2.5 groups than in Con group. Protein efficiency ratio was significantly higher in Cb1.0 and Cb1.5 groups than in Con group. Dietary vitamin B12 supplementation significantly increased hemocyte counts, non-specific immune responses and antioxidant enzyme activity of shrimp. Hepatopancreas amylase activity was significantly increased in Cb1.0 group than in Con group. Intestinal histomorphology was not significantly affected, while hepatic and muscle vitamin B12 levels were increased in a significant linear trend. The expression of growth, vitamin B12 metabolism and immune-related genes were significantly upregulated by dietary vitamin B12 supplementation. Based on polynomial regression analysis of WG to dietary vitamin B12 levels, the optimal dietary vitamin B12 requirement seems to be 1.48 mg/kg for Pacific white shrimp.