REG3γ is considered to have a protective role against infection with Gram-positive bacteria due to its bactericidal activity, but evidence from in vivo studies is lacking. We generated a REG3γ(-/-) mouse, and investigated the effect of lack of REG3γ on intestinal mucus distribution, spatial compartmentalization of bacteria, and expression of innate immunity genes. Infection studies were also performed with Gram-positive and Gram-negative pathogens to investigate the antimicrobial role of REG3γ. REG3γ(-/-) mice display altered mucus distribution, increased bacterial contact with the epithelium, and elevated inflammatory markers in the ileum without histological evidence of pathology. Infection response pathway genes were differentially expressed in both Listeria monocytogenes and Salmonella enteritidis infected REG3γ(-/-) and wild-type (wt) mice. Higher amounts of myeloperoxidase and interleukin-22 transcripts were present in the ileal mucosa of REG3γ(-/-) than wt mice, but translocation to the organs was unaffected. We concluded that REG3γ has a protective role against mucosal infection with pathogenic Listeria and Salmonella in vivo. REG3γ is equally distributed throughout the mucus and its absence results in increased epithelial contact with the microbiota resulting in low-grade inflammation. REG3γ can bind to Gram-negative and Gram-positive bacteria and influence mucus distribution in the ileum, properties which may contribute to mucosal protection.