The application of probiotics for disease control in aquaculture is now a convincing approach towards replacement of antibiotics, which can cause adverse effects in aquatic animals and humans. In this study, we combined 2 probiotics, Lactobacillus acidophilus and Saccharomyces cerevisiae, with shrimp feed to create 2 formulas (WU8 and WU9), which were fed for 10 d to juvenile shrimp Penaeus vannamei. The shrimps were then subjected to a challenge infection with Vibrio parahaemolyticus, the causative agent of acute hepatopancreas necrosis disease (AHPND). The protective effects of probiotics against bacterial infection were investigated through histopathology of the hepatopancrease and immunological evaluation of shrimp. Both WU8 and WU9 probiotic mixtures (1:1, at 108 and 109 CFU kg diet-1) increased blasenzellen hepatopancreatic epithelial cells and reduced pathology caused by AHPND. After 10 d of feeding, hemocyte parameters, including the total hemocyte count, percent of granular hemocytes, and phenoloxidase activity, increased significantly and were still increasing at 24 h post infection. Crustin and penaeidin 3 genes were also highly upregulated in hemocytes before and after 24 h of bacterial challenge and significantly upregulated in the hepatopancreas 1 to 5 d post-infection. A significantly higher survival rate was observed in shrimp fed with the probiotic supplemented diet (>90%) in comparison to the control group (60%). In conclusion, probiotic mixtures of L. acidophilus and S. cerevisiae reduced hepatopancreas pathology and protected shrimp from a challenge with AHPND.