The present study aimed to evaluate the effects of dietary inositol level on the growth performance and barriers function of hemolymph and intestine of Litopenaeus vannamei. Six isonitrogenous and isolipidic diets were formulated with analyzed inositol levels being 0.86 (basal diet), 1.34, 1.40, 2.30, 3.88, and 7.50 g kg−1, respectively. The results indicated that appropriate dietary inositol could improve the growth performance of Litopenaeus vannamei. The optimal dietary inositol requirement was 1.44 g kg−1 according to broken-line regression analysis with the WGR as the evaluation index. The activities of non-specific immune enzymes (SOD, ACP, ALP and NOS) in hemolymph and the expression levels of genes involved in the Toll signaling pathway (toll, pelle and traf6) in hemocyte of shrimp fed diet containing 1.40 g kg−1 inositol significantly higher than those in shrimp fed diet containing 0.86 g kg−1 inositol, and the activities of PO and SAHR in hemolymph and the expression levels of genes involved in immune enzymes (Mn-sod, Cu/Zn sod, acp, akp and po) in hemocyte of shrimp fed diet containing 1.40, 2.30, 3.88 and 7.50 g kg−1 inositol significantly higher than those in shrimp fed diet containing 0.86 g kg−1 inositol, suggesting that appropriate dietary inositol can improve hemolymph immunity by increasing the activities of antioxidant and immune enzymes in the hemolymph. In addition, the intestinal VH and the expression levels of genes involved in intestinal health (zo-1, cyclC, tgfβ and il2) in shrimp fed diet containing 1.40 g kg−1 inositol were the highest. Meanwhile, the inositol content, VW, and the expression levels of genes involved in the IMD pathway (imd) and anti-lipopolysaccharide factor (alf) in the intestine were significantly correlated with dietary inositol level. These results suggested that appropriate inositol can improve intestinal health by improving intestinal morphology and the expression of health-related genes. Although dietary inositol had no significant effect on the microbial composition spectrum diversity in the hemolymph and intestine of L. vannamei, it contributed to increasing the abundance of beneficial bacteria and decreasing the abundance of opportunistic pathogens. KEGG analysis showed that dietary inositol level had significantly positive effects on multiple immune and metabolic pathways. In conclusion, dietary appropriate inositol enhanced hemolymph homeostasis and host immune defense by improving the barrier function of immune and microbial in hemolymph and improved the intestine health status by enhancing the barrier function of physical, immune and microbial in intestine.