In this study, we investigated the impact of β-1,3-glucan on the immune responses and gut microbiota of the river prawn (Macrobrachium nipponense) in the presence of Vibrio parahaemolyticus stress. Shrimps were fed one of the following diets: control (G1), 0.2% curdlan (G2), 0.1% β-1,3-glucan (G3), 0.2% β-1,3-glucan (G4), or 1.0% β-1,3-glucan (G5) for 6 weeks and then challenged with V. parahaemolyticus for 96 h. Under Vibrio stress, shrimps in G4 exhibited the highest length gain rate, weight gain rate, and survival rate. They also showed increased intestinal muscle thickness and villus thickness compared to the control and 0.2% curdlan groups. The apoptosis rate was lower in G4 than in the control group, and the digestive enzyme activities (pepsin, trypsin, amylase, and lipase), immune enzyme activities (acid phosphatase, alkaline phosphatase, lysozyme, and phenoxidase), and energy metabolism (triglyceride, cholesterol, glycogen, and lactate dehydrogenase) were enhanced. Expression levels of growth-related genes (ecdysone receptor, calmodulin-dependent protein kinase I, chitin synthase, and retinoid X receptor) and immune-related genes (toll-like receptor 3, myeloid differentiation primary response 88, mitogen-activated protein kinase 7, and mitogen-activated protein kinase 14) were higher in G4 than in the control. Microbiota analysis indicated higher bacterial abundance in shrimps fed β-1,3-glucan, as evidenced by Sob, Chao1, and ACE indices. Moreover, 0.2% β-1,3-glucan increased the relative abundances of Bacteroidota and Firmicutes while reducing those of Corynebacteriales and Lactobacillales. In summary, β-1,3-glucan enhances immune enzyme activities, alters immune-related gene expression, and impacts gut microbial diversity in shrimp. These findings provide valuable insights into the mechanisms underlying β-1,3 glucan's immune-enhancing effects.