Abstract: To comprehensively evaluate the effects of dietary supplementation with Bacillus subtilis (BS) on the growth, immunity, and resistance to Vibrio parahaemolyticus infection in Litopenaeus vannamei, 4500 shrimp (0.95±0.25) g were randomly assigned to five groups, including a control group without BS, experimental groups supplemented with BS at 0.2%, 0.4%, and 0.8%, and a positive control group with 0.05% Sanhuang Powder (SH). A 42-day feeding trial and a 48-hour V. parahaemolyticus challenge were conducted. Results showed that BS and SH supplementation significantly improved body length, body weight, specific growth rate, weight gain rate, and feed conversion ratio. Compared with the control group, the 0.4% BS group exhibited significantly reduced activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in hemolymph, while the 0.8%BS group showed significantly increased superoxide dismutase (SOD) content and total antioxidant capacity (T-AOC) in hemolymph and hepatopancreas. The 0.05%SH group exhibited significantly enhanced acid phosphatase (ACP) and T-AOC in hemolymph and SOD content in hepatopancreas, while the activities of ALT decreased significantly. Furthermore, BS and SH supplementation significantly upregulated the expression of immune-related factors such as Hsp70, LGBP, β-GBP-HDL, Crustin, and Lysozyme in the hepatopancreas after V. parahaemolyticus infection and improved the survival rate of shrimp. Specifically, the survival rates in the 0.2%, 0.4%, and 0.8%BS, and 0.05%SH groups increased from 43.3% (control) to 66.7%, 76.7%, 73.3%, and 80.0%, respectively. This study demonstrates that dietary supplementation with appropriate amount of BS enhance the growth performance, nonspecific immunity, and hepatopancreas immune gene expression in L. vannamei, thereby improving resistance to V. parahaemolyticus infection.