Crustins are a kind of antimicrobial peptide (AMP) that exist in crustaceans. Some crustins do not have direct antimicrobial activity but exhibit in vivo defense functions against Vibrio . However, the underlying molecular mechanism is not clear. Here, the regulatory mechanism was partially revealed along with the characterization of the immune function of a type I crustin, LvCrustin I -2, from Litopenaeus vannamei . LvCrustin I -2 was mainly detected in hemocytes, intestines and gills and was apparently up-regulated after Vibrio parahaemolyticus infection. Although the recombinant LvCrustin I-2 protein possessed neither antibacterial activity nor agglutinating activity, the knockdown of LvCrustin I -2 accelerated the in vivo proliferation of V. parahaemolyticus . Microbiome analysis showed that the balance of intestinal microbiota was impaired after LvCrustin I -2 knockdown. Further transcriptome analysis showed that the intestinal epithelial barrier and immune function were impaired in shrimp after LvCrustin I -2 knockdown. After removing the intestinal bacteria via antibiotic treatment, the phenomenon of impaired intestinal epithelial barrier and immune function disappeared in shrimp after LvCrustin I -2 knockdown. This indicated that the impairment of the shrimp intestine after LvCrustin I -2 knockdown was caused by the dysbiosis of the intestinal microbiota. The present data suggest that crustins could resist pathogen infection through regulating the intestinal microbiota balance, which provides new insights into the functional mechanisms of antimicrobial peptides during pathogen infection.