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The aim of this study was to determine the effects of Pro-A (a mixture of Bacillus subtilis, B. licheniformis, B. pumilus) on the growth performance, feed utilization, immune responses, and intestinal microbiota of whiteleg shrimp (Litopenaeus vannamei). The experiment was conducted with four iso-nitrogenous (45.4% crude protein) and iso-caloric (19.9 KJ g−1) diets supplemented with different levels (0%, 0.05%, 0.1%, and 0.2%) of Pro-A. Whiteleg shrimp (n = 1400, average body weight: 0.45 g) were randomly assigned to 500-L composite tanks (filled with 400 L of brackish water) at a stocking rate of 70 shrimp tank−1 for 10 weeks. There were five replicates per dietary treatment. The results showed that the diet containing 0.1% Pro-A significantly increased the growth, protein efficiency ratio, feed conversion ratio, and survival rate compared to the control. Diets containing 0.05% and 0.1% Pro-A significantly increased the digestive enzymes compared to the control. Furthermore, diet containing 0.1% Pro-A significantly improved hematological and immune profiles. The total vibrio count was lowest in the diet supplemented with 0.1% of Pro-A, accordingly, the bacterial clearance efficiency was higher in this diet. The whiteleg shrimp fed the Pro-A diets (at 0.05%, 0.1%, and 0.2%) had lower cumulative mortality than the control after being challenged with Vibrio parahaemolyticus. Our results suggest that dietary supplementation with 0.1% Pro-A is the most effective in improving the performance and immunity of whiteleg shrimp; however, this should be confirmed in a further study under full-scale commercial trials.