<p>This study evaluated the dietary vitamin B₁₂ (cyanocobalamin) requirement of Pacific white shrimp (<i>Penaeus vannamei</i>) post-larvae for growth, feed utilization, innate immunity, antioxidant capacity, and vitamin B₁₂ metabolism. Six casein-based semi-purified diets (Con, C0.5, C1.0, C2.0, C4.0, and C8.0) were prepared by adding cyanocobalamin at 0.00, 0.50, 1.00, 2.00, 4.00, and 8.00&#xa0;mg/kg levels. Analyzed vitamin B₁₂ levels in diets were 0.10, 0.53, 1.10, 2.17, 4.78, and 8.35&#xa0;mg/kg, respectively. Triplicate groups per treatment, each containing 100 post-larvae (0.9 ± 0.1&#xa0;mg) per tank, were fed the diets for 30&#xa0;days. The growth performance and feed efficiency were significantly increased in C2.0, C4.0, and C8.0 groups compared to the Con group, and significantly higher survival was observed in the C2.0 group than in the control. Vitamin B₁₂ supplementation significantly increased total hemocyte and hyaline cell counts in shrimp hemolymph. Hemolymph and hepatopancreas phenoloxidase, lysozyme, and catalase activities were significantly affected by dietary vitamin B₁₂ supplementation. Hemolymph glutathione peroxidase activity was significantly higher in C4.0 and C8.0 groups than in the Con group. Hepatopancreas nitroblue tetrazolium activity and gene expressions of target of rapamycin, insulin-like growth factor-1, crustin, penaeidin, prophenoloxidase, methylmalonyl-CoA mutase mitochondrial, and methionine synthase were significantly upregulated with vitamin B₁₂ supplementation. Whole-body vitamin B₁₂ levels were increased linearly, corresponding to graded dietary vitamin B₁₂ levels. Broken-line regression analysis indicated that the optimum dietary vitamin B₁₂ requirement for maximum growth of Pacific white shrimp post-larvae is 2.13&#xa0;mg/kg.</p>