Water manganese (Mn) is apparently insufficient to meet aquatic animals' metabolic requirement, and their food is considered as the major Mn source. Currently, researches reporting the effects of dietary Mn inclusion on physiological and metabolic function in Litopenaeus vannamei (L. vannamei) are very scarce. Therefore, 8-week feeding trail was conducted to determine dietary Mn requirement for L. vannamei and further evaluate the effects of dietary Mn supplementation on tissue Mn concentration, antioxidant enzyme activity, nonspecific immune enzymes activity and intestinal microbiota composition. Our results demonstrated that the optimum dietary Mn requirement for L. vannamei was determined to be 44.83 mg/kg based on the per cent weight gain; supplementing 39.57 mg/kg, 54.57 mg/kg and 72.67 mg/ kg Mn significantly increased (p < .05) the activity of serum lysozyme (LZM), nitric oxide synthase and alkaline phosphatase, the activity of antioxidative capacity and glutathione in serum and hepatopancreas of L. vannamei. Supplementing 111.97 mg/ kg Mn significantly increased (p < .05) hepatopancreas Mn concentration and serum LZM activity. Intestinal microbiota analysis showed that supplementing 39.57 mg/kg and 111.97 mg/kg Mn significantly influenced (p < .05) intestinal microbiota composition and function. These findings might help provide a scientific basis for the commercial application of Mn in L. vannamei.