Of human pathogenic Vibrio species, V. mimicus causes gastroenteritis whereas V. vulnificus causes fatal septicemia after consumption of contaminated seafood. These two pathogens produce hemolytic toxins termed V. mimicus hemolysin (VMH) and V. vulnificus hemolysin (VVH), respectively. These toxins elicit the cytolysis of various eukaryotic cells, as well as erythrocytes. The human intestine secretes cationic antimicrobial peptides (AMPs) to prevent infectious diseases. Paneth cells in the small intestine secrete α-defensin 5 (HD-5) and epithelial cells in the large intestine produce LL-37. In the present study, we examined the bactericidal activities of AMPs against V. mimicus and V. vulnificus. Although HD-5 showed no bactericidal activity, LL-37 revealed significant activity against both Vibrio species, suggesting that neither V. mimicus nor V. vulnificus can multiply in the large intestine. We also tested whether AMPs had the ability to inactivate the hemolytic toxins. Only HD-5 was found to inactivate VMH, but not VVH, in a dose-dependent manner through the direct binding to VMH. Therefore, it is considered that V. mimicus cannot penetrate the small intestinal epithelium because the cytolytic action of VMH is inactivated by HD-5.