Antimicrobial peptides (AMPs), produced by several species including bacteria, insects, amphibians and mammals as well as by chemical synthesis and genetically engineered microorganisms, are of great importance in maintaining normal gut homeostasis. AMPs exhibit a broad spectrum of antimicrobial activity and inhibit microbial cells by interaction with their membranes or by other mechanisms, such as inhibition of cell-wall synthesis or suppression of nucleic acid or protein synthesis. In addition to their direct antimicrobial functions, they have multiple roles in the stabilization of epithelial barrier integrity and function as potent immune regulators. The fate of AMPs in vivo is poorly understood, prompting the need for studying AMPs pharmacokinetics. This review summarizes the current knowledge about the basic biology of AMPs and discusses the features of AMPs in gut homeostasis and their relative mechanisms of action.