The mammalian intestine hosts a complex community of beneficial bacteria that are maintained in the lumen with minimal contact with mucosal surfaces. Microbial colonization of germ-free mice triggers the expression of RegIIIγ, a secreted C-type lectin that binds intestinal bacteria and has direct antimicrobial activity. RegIIIγ and its human counterpart, HIP/PAP, are directly antimicrobial proteins that bind bacterial targets via interactions with peptidoglycan carbohydrate. These proteins represent an evolutionarily primitive form of lectin-mediated innate immunity and reveal intestinal strategies for maintaining symbiotic host-microbial relationships. The human gut is home to a vast consortium of symbiotic bacteria that metabolize dietary substances, such as plant polysaccharides, that are otherwise indigestible by their human hosts. Indigenous gut microbes make essential contributions to human nutrient metabolism and, in return, inhabit a protected, nutrient-rich environment. Maintaining the mutually beneficial nature of this relationship requires strict sequestration of resident bacteria in the intestinal lumen, as microbial incursions across epithelia can elicit inflammation and sepsis. Epithelial antimicrobial proteins are evolutionarily ancient innate immune effectors. As key elements of intestinal mucosal defense, they likely play an important role in maintaining mutually beneficial host-microbial relationships by restricting contact between resident microbes and mucosal surfaces. This idea is underscored by the fact that deficiencies in antimicrobial peptide expression are associated with inflammatory bowel disease (IBD), a chronic inflammatory disorder thought to be triggered by resident gut microbes. However, although cationic antimicrobial peptides such as defensins are well-characterized, the full repertoire of gut antimicrobial mechanisms remains undefined. Here we show that resident gut bacteria drive intestinal epithelial expression of a C-type lectin that binds peptidoglycan and has direct antimicrobial activity, revealing a primitive mechanism of lectin-mediated innate immunity. RegIIIγ and HIP/PAP are peptidoglycan-binding proteins with direct antibacterial activity. Immunogold electron microscopy revealed that RegIIIγ is present in Paneth cell secretory granules. Granule contents are released apically, indicating that RegIIIγ is targeted to the gut lumen, which harbors large resident bacterial populations. RegIIIγ binds to Gram-positive bacteria, as shown by flow cytometry and dual staining with fluorochrome-conjugated RegIIIγ and WGA. Pull-down assays confirmed that RegIIIγ binds peptidoglycan, a molecule exposed on the Gram-positive bacterial surface. RegIIIγ and HIP/PAP bind to chitin and mannose, but not dextran or monomeric GlcNAc. These results suggest that RegIIIγ and HIP/PAP are pattern-recognition proteins that recognize the microbe-associated molecular pattern represented by the extended glycan chains of peptidoglycan. RegIIIγ and HIP/PAP have antibacterial activity against Gram-positive bacteria. They inhibit the viabilityThe mammalian intestine hosts a complex community of beneficial bacteria that are maintained in the lumen with minimal contact with mucosal surfaces. Microbial colonization of germ-free mice triggers the expression of RegIIIγ, a secreted C-type lectin that binds intestinal bacteria and has direct antimicrobial activity. RegIIIγ and its human counterpart, HIP/PAP, are directly antimicrobial proteins that bind bacterial targets via interactions with peptidoglycan carbohydrate. These proteins represent an evolutionarily primitive form of lectin-mediated innate immunity and reveal intestinal strategies for maintaining symbiotic host-microbial relationships. The human gut is home to a vast consortium of symbiotic bacteria that metabolize dietary substances, such as plant polysaccharides, that are otherwise indigestible by their human hosts. Indigenous gut microbes make essential contributions to human nutrient metabolism and, in return, inhabit a protected, nutrient-rich environment. Maintaining the mutually beneficial nature of this relationship requires strict sequestration of resident bacteria in the intestinal lumen, as microbial incursions across epithelia can elicit inflammation and sepsis. Epithelial antimicrobial proteins are evolutionarily ancient innate immune effectors. As key elements of intestinal mucosal defense, they likely play an important role in maintaining mutually beneficial host-microbial relationships by restricting contact between resident microbes and mucosal surfaces. This idea is underscored by the fact that deficiencies in antimicrobial peptide expression are associated with inflammatory bowel disease (IBD), a chronic inflammatory disorder thought to be triggered by resident gut microbes. However, although cationic antimicrobial peptides such as defensins are well-characterized, the full repertoire of gut antimicrobial mechanisms remains undefined. Here we show that resident gut bacteria drive intestinal epithelial expression of a C-type lectin that binds peptidoglycan and has direct antimicrobial activity, revealing a primitive mechanism of lectin-mediated innate immunity. RegIIIγ and HIP/PAP are peptidoglycan-binding proteins with direct antibacterial activity. Immunogold electron microscopy revealed that RegIIIγ is present in Paneth cell secretory granules. Granule contents are released apically, indicating that RegIIIγ is targeted to the gut lumen, which harbors large resident bacterial populations. RegIIIγ binds to Gram-positive bacteria, as shown by flow cytometry and dual staining with fluorochrome-conjugated RegIIIγ and WGA. Pull-down assays confirmed that RegIIIγ binds peptidoglycan, a molecule exposed on the Gram-positive bacterial surface. RegIIIγ and HIP/PAP bind to chitin and mannose, but not dextran or monomeric GlcNAc. These results suggest that RegIIIγ and HIP/PAP are pattern-recognition proteins that recognize the microbe-associated molecular pattern represented by the extended glycan chains of peptidoglycan. RegIIIγ and HIP/PAP have antibacterial activity against Gram-positive bacteria. They inhibit the viability