The single WAP domain-containing protein (SWD) is a type III crustin antimicrobial peptide whose function is to defense the host animal against the bacterial infection by means of antimicrobial and antiproteinase activities. A study of SWD from Litopenaeus vannamei (LvSWD) is reported herein about its activities and function against bacteria, particularly the AHPND-inducing Vibrio parahaemolyticus (VPAHPND) that causes acute hepatopancreatic necrosis disease (AHPND). The LvSWD is mainly synthesized in hemocytes and up-regulated in response to VPAHPND infection. Over-expressed mature recombinant LvSWD (rLvSWD) and its WAP domain (rLvSWD-WAP) are able to strongly inhibit subtilisin but not trypsin, chymotrypsin and elastase. The rLvSWD inhibits subtilisin with the inhibition constant (Ki) of 14.3 nM. However, only rLvSWD exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Unlike the rLvSWD, the rLvSWD-WAP does not possess antimicrobial activity. Therefore, the killing effect of rLvSWD on VPAHPND and Bacillus megaterium was studied. The MIC of 30 μM against VPAHPND is bactericidal whereas the MIC against B. megaterium is not. With four times the MIC of rLvSWD, the VPAHPND-treated post larval shrimp are able to survive longer with 50% survival rate as long as 78 h as compared to 36 h of the infected shrimp without rLvSWD. The antimicrobial activity of LvSWD against the VPAHPND infection suggests its potential application for disease control in aquaculture.