The mammalian gastrointestinal (GI) tract is host to a complex community including microbes and immune cells that converge at the epithelial barrier. Despite nearly constant interaction between these two populations, homeostasis is largely maintained. This is accomplished, in part, through non- inflammatory barrier host defense mechanisms, many of which have yet to be explored. Using a well- established model system of non-inflammatory commensal colonization with Enterococcus faecalis (EF), we confirmed a significant increase in complement component 3 (C3) gene expression in the epithelium of the distal small intestine (DSI). Complement has been associated with intestinal cells in more recent years, but C3 gene expression in the DSI in the absence of inflammatory stimuli has not been observed until now. Using state of the art spatial transcriptomics, we characterized clusters of cells in the DSI that were responsible for the increase in C3 gene expression in the DSI and found that they were made up of enterocytes, Paneth cells and fibroblasts. We also found that C3 protein in both DSI tissue and luminal contents were present during homeostasis and DSI luminal content C3 increased in response to EF colonization. Within the DSI luminal contents, we showed that commensal bacteria are bound by C3. Because we found novel cellular expression of C3 in the DSI during commensal colonization, we hypothesize that C3 produced by gut epithelium is secreted into the lumen to mediate non-inflammatory commensal-host interaction. To characterize the relevance of these sources and the role of C3 in the DSI, we propose two aims. Aim 1 will characterize the cellular sources of intestinal C3 and the specificity of C3 bacterial binding in the DSI lumen. Aim 2 will investigate the function of C3 in the DSI to facilitate host-microbe homeostasis. Completion of these aims will drive future mechanistic studies of C3 in the DSI, and its contribution to non-inflammatory host defense.