Objective: Reg3g has been proposed to have a protective role against infection due to its bactericidal effect on Gram-positive bacteria, but evidence from in vivo studies is lacking. Therefore we generated a Reg3g-/- mouse, to determine its role in intestinal homeostasis and protection against experimental infection. Methods: Reg3g-/- mice were phenotyped using histological methods and a range of innate and immune markers. To investigate the antimicrobial role of Reg3g we experimentally infected mice with Gram-positive Listeria monocytogenes and Gram-negative Salmonella entertitidis and measured translocated bacteria, mucosal and systemic markers of infection. Results: Reg3g-/- mice display altered ileal mucus distribution and increased bacterial contact with the epithelium. , concomitant with This increased the inflammatory status in of the ileal mucosa and increased expression of Il-22, myeloperoxidase (MPO) and serum chemokines in serum. In response to infection, Reg3g-/- mice showed transcriptome changes and elevated levels of mucosal MPO in the ileum, but no increased bacterial translocation to the organs. Conclusions: Reg3g is equally distributed throughout the mucus of wild type (wt) mice and its absence results in an altered distribution of the ileal mucus. Reg3g deficiency also results resulted in increased bacterial contact with the epithelium and heightened inflammatory responses in the ileal mucosa. We propose that Reg3g binds pathogens suggesting it and contributes to mucus barrier function by ensnaring bacteria. Compared to wt mice, Reg3g-/- mice infected with S. enteritidis and L. monocytogenes show an increase of mucosal inflammatory markers indicating the protective, anti-microbial roles of Reg3g in defence against both Gram-positive and -negative bacteria.