Microbial drug resistance is becoming increasingly severe due to antibiotic abuse. The development and utilization of antimicrobial peptides is one of the important ways to solve this difficult problem. Crustins are a family of antimicrobial peptides that play important roles in the innate immune system of crustaceans. Several types of crustins exist in shrimp and their activities vary greatly. In the present study, we studied the immune function of one newly identified crustin and found that the type VI crustin encoding gene in Litopenaeus vannamei (LvCrustinVI) was mainly expressed in gills. Its expression was significantly up-regulated after Vibrio parahaemolyticus infection and knockdown of the gene promoted Vibrio proliferation in the hepatopancreas of shrimp, indicating that LvCrustinVI was involved in pathogens infection. The recombinant LvCrustinVI (rLvCrustinVI) showed strong inhibitory activities against both Gram-negative and Gram-positive bacteria, and exhibited binding activities with the bacteria and bacterial polysaccharides including Glu, LPS and PGN. In the presence of Ca, rLvCrustinVI showed a strong agglutination effect on V. parahaemolyticus and could significantly enhance the phagocytic ability of shrimp hemocytes against V. parahaemolyticus. In conclusion, LvCrustinVI played important roles as antimicrobial peptide and opsonin in the innate immune defense of L. vannamei. The study enriched our understanding of the functional activity of Crustin and provides an important basis for the development and utilization of antimicrobial peptides.