Abstract Impaired expression of α-defensin antimicrobial peptides and overproduction of the proinflammatory cytokine IL-1β have been associated with inflammatory bowel disease. In this study, we examine the interactions between α-defensins and IL-1β and the role of defensin deficiency in the pathogenesis of inflammatory bowel disease. It was found that matrix metalloproteinase-7-deficient (MMP-7−/−) mice, which produce procryptdins but not mature cryptdins (α-defensins) in the intestine, were more susceptible to dextran sulfate sodium-induced colitis. Furthermore, both baseline and dextran sulfate sodium-induced IL-1β production in the intestine were significantly up-regulated in MMP-7−/− mice compared with that in control C57BL/6 mice. To elucidate the molecular mechanism for the increased IL-1β production in defensin deficiency in vivo, we evaluated the effect of defensins on IL-1β posttranslational processing and release. It was found that α-defensins, including mouse Paneth cell defensins cryptdin-3 and cryptdin-4, human neutrophil defensin HNP-1, and human Paneth cell defensin HD-5, blocked the release of IL-1β from LPS-activated monocytes, whereas TNF-α expression and release were not affected. Unlike α-defensins, human β-defensins and mouse procryptdins do not have any effect on IL-1β processing and release. Thus, α-defensins may play an important role in intestinal homeostasis by controlling the production of IL-1β.