Radiation-induced enteritis, a significant concern in abdominal radiation therapy, is associated closely with gut microbiota dysbiosis. The critical mucus layer plays a pivotal role in preventing the translocation of commensal and pathogenic microbes. Although the significant expression of REGγ in intestinal epithelial cells is well established, its role in modulating the mucus layer and gut microbiota remains enigmatic. The current study revealed notable changes in gut microorganisms and metabolites in irradiated mice lacking REGγ, as opposed to wild-type mice. Concomitant with gut microbiota dysbiosis, REGγ deficiency facilitated the infiltration of neutrophils and macrophages, thereby exacerbating intestinal inflammation after irradiation. Furthermore, fluorescence in situ hybridization assays unveiled an augmented proximity of bacteria to intestinal epithelial cells in REGγ knockout mice after irradiation. Mechanistically, deficiency of REGγ led to diminished goblet cell populations and reduced expression of key goblet cell markers, Muc2 and Tff3, observed in both murine models, minigut organoid systems and human intestinal goblet cells, indicating the intrinsic role of REGγ within goblet cells. Interestingly, although administration of broad-spectrum antibiotics did not impact the alteration of goblet cell numbers and MUC2 secretion, it effectively attenuated inflammation levels in the ileum of irradiated REGγ absent mice, aligning them with their wild-type counterparts. Collectively, these findings highlight the crucial contribution of REGγ in counteracting radiation-triggered microbial imbalances and cell-autonomous regulation of mucin secretion.