BACKGROUND & AIMS: Regenerating islet-derived protein (REG3 gamma), an antimicrobial peptide, typically expressed by intestinal epithelial cells (IEC), plays crucial roles in intestinal homeostasis and controlling gut microbiota. However, the mechanisms that regulate IEC expression of REG3 gamma are still largely unclear. In this study, we investigated whether and how interleukin (IL) 33, an alarmin produced by IEC in response to injury, regulates REG3 gamma expression in IEC, thus contributing to intestinal homeostasis. METHODS: IEC were isolated from wild-type and IL33(-/-) mice to determine expression of REG3 gamma and other antimicrobial peptides by quantitative real-time polymerase chain reaction and Western blot. IEC cell lines were used for mechanistic studies. 16S rRNA pyrosequencing analysis was used for measuring gut microbiota. Citrobacter rodentium was used for enteric infections. RESULTS: The expression of REG3 gamma, but not ,beta-defensins, in IECs of IL33(-/-) mice was significantly lower than wild-type mice. IL33 treatment induced IEC expression of REG3 gamma in both mice and human cell lines. Mechanistically, IL33 activated STAT3, mTOR, and ERK1/2 in IEC. Inhibition of these pathways abrogated IL33-induction of REG3 gamma. IL33(-/-) mice demonstrated higher bacteria loads and altered microbiota composition. IL33 did not directly inhibit bacterial growth, but promoted wild-type, not REG3 gamma KO, IECs to kill bacteria in vitro. Consistently, C rodentium infection induced IEC IL33 expression, and IL33(-/-) mice demonstrated an impaired bacterial clearance with C rodentium infection. CONCLUSIONS: Our study demonstrated that IL33, which is produced by IEC in response to injury and inflammatory stimulation, in turn promotes IEC expression of REG3 gamma, and controls the gut microbiota of the host.