The single and synergistic effects of dietary arabinoxylan-oligosaccharide (AXOS) and inulin at different doses (2, 4, and 8 mg/g diet) on survival, growth performance, gut microbiota, and immune response in Pacific white shrimp Litopenaeus vannamei were assessed. Singular application of either inulin or AXOS at doses of 4 mg/g diet showed the most stimulatory effects on the growth rate and gene expression levels of chitinase, cathepsin L, chymotrypsin, and extracellular signal-regulated kinase (ERK) in each single prebiotic feeding trial. Compared with single prebiotic treatments, simultaneous application of AXOS and inulin at 4 mg/g diet enhanced remarkably the growth parameters of shrimp and the expression of related genes (chitinase, cathepsin L, chymotrypsin, ERK, myeloid differentiation factor 88, and phenoloxidase) after 8-week feeding ( P <0.05). Additionally, gut microbiota analysis indicated the dietary supplementation with combined prebiotics increased significantly the bacterial community richness and relative abundance of Bacillus, Pseudomonas , Bacteriovorax, and Lactobacillus, and reduced the abundance of Vibrio, Rhodococcus, and Photobacterium in the digestive tract of L . vannamei. Compared with the single prebiotic treatment and the control, combined prebiotics supplementation boosted notably the survival rate and expression levels of immune-related genes in shrimp infected with Vibrio alginolyticus or white spot syndrome virus. Therefore, simultaneous application of AXOS and inulin shall have a great potential of dietary supplement in the culture of L . vannamei.