The use of probiotics in animal husbandry is an alternative solution to promote animal health and disease prevention and becomes more and more popular. In addition, Bacillus is amongst the most common used and intensively studied as probiotic in aquaculture. Although, the precise mechanisms of Bacillus probiotic action are not well documented and remain largely unknown. The abundant of microbiota which presents naturally in the culture system or test animal often interferes the results. Here, we aim to provide a comprehensive understanding about interaction among probiotic Bacillus subtilis DSM33018, the host-brine shrimp Artemia franciscana larvae, and the pathogen Vibrio campbellii in a gnotobiotic system. The strain DSM33018 significantly increased the survival of brine shrimp challenged with Vibrio campbelii (survival rate of 77±8% with Bacillus supplement compared to 24±2% without Bacillus supplement). No growth suppression of V.campbellii in the cultured water was observed, although Bacillus reduced the amount of attached Vibrio in Artemia intestinal tract (1.2x105 cells/20animals in Bacillus treatment compared to 3.4x106 cells/20animals in treatment without Bacillus). Interestingly, mRNA expression of gene encoding antioxidant enzyme Superoxidase dismutase (SOD), antimicrobial enzyme Phenoloxidase (ProPO) increased significantly in treatment with Bacillus supplementation. We also found out that Bacillus administration triggered some shrimp innate related genes leading to improve the early innate responses of the host. In conclusion, our findings showed that Bacillus protected the brine shrimp from Vibrio infection by reducing the attached V.campbellii in the shrimp intestinal tract and enhancing shrimp innate immunity, especially antioxidant capacity for Vibriosis resistance.