The mammalian intestine is home to similar to 100 trillion bacteria that perform important metabolic functions for their hosts. The proximity of vast numbers of bacteria to host intestinal tissues raises the question of how symbiotic host-bacterial relationships are maintained without eliciting potentially harmful immune responses. Here, we show that RegIII gamma, a secreted antibacterial lectin, is essential for maintaining a similar to 50-micrometer zone that physically separates the microbiota from the small intestinal epithelial surface. Loss of host-bacterial segregation in RegIII gamma(-/-) mice was coupled to increased bacterial colonization of the intestinal epithelial surface and enhanced activation of intestinal adaptive immune responses by the microbiota. Together, our findings reveal that RegIII gamma is a fundamental immune mechanism that promotes host-bacterial mutualism by regulating the spatial relationships between microbiota and host.