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Epidermal growth factor receptor (EGFR) is a key regulator of epithelial proliferation, survival and migration in the colon, where it functions to stimulate epithelial repair following injury. However, recent work by our lab has uncovered a novel role for epithelial EGFR to limit inflammation and subsequent tumorigenesis in mouse colitis models. This anti-inflammatory role for EGFR suggested that inhibition of epithelial innate immune responses might be a key mechanism underlying the therapeutic potential for EGF in IBD. In this study, our aim was to determine how EGFR regulated the epithelial cell response to lipopolysaccharide (LPS), and how a novel target of the EGFR kinase, coiled-coil domain containing 50 (CCDC50), mediates this response. EGFR inhibits colon epithelial responses to LPS by blocking NFκB activation through the adapter protein CCDC50. YAMC mouse colon epithelial (MCE) cells or human HT-29 cells were stimulated with LPS with or without EGF co-treatment. NFκB activity was measured in YAMC cells (NF-YAMC) stably transfected with an NFκB luciferase reporter. IL-8 and CXCL2 mRNA were measured by qPCR, and CXCL2 secretion was measured by ELISA. CCDC50 or TNFAIP3 were knocked down by siRNA with non-targeting siRNA as control. Knockdown was confirmed by qPCR. LPS induced concentration-dependent NFκB activity (EC50 = 40 ng/mL) in NF-YAMC cells, which was inhibited with escalating concentrations of EGF (see figure; IC50 = 0.2 ng/mL EGF, n = 6). This inhibitory effect of EGF was blocked with an EGFR inhibitor, and in MCE cells expressing a dominant-negative EGFR (EGFRwa2). EGF alone did not affect basal NFκB activity. This response was not specific for LPS, as EGF also inhibited NFκB activity induced by L18-MDP (a cell-permeable muramyl dipeptide analog). We next determined that EGF inhibited epithelial chemokine production following LPS stimulation. EGF inhibited LPS-stimulated induction of mRNA for IL-8 (HT-29 cells) and CXCL2 (YAMC and HT-29 cells), and protein for CXCL2 (YAMC cells). Using immunoprecipitation and Western blot, we determined that EGF induced tyrosine phosphorylation of CCDC50 in YAMC cells. Furthermore, siRNA-mediated CCDC50 knockdown blocked the ability of EGF to inhibit LPS-induced NFκB activity and CXCL2 mRNA expression, compared to non-targeting siRNA control. Since CCDC50 has previously been shown to interact with TNFAIP3 (A20), we determined whether CCDC50 required TNFAIP3 to inhibit LPS-induced NFκB activity by siRNA-mediated TNFAIP3 knockdown. However, loss of TNFAIP3 did not prevent the inhibitory effect of EGF, suggesting that the EGFR/CCDC50 mechanism involves an alternate pathway for NFκB inhibition. The inhibition of LPS-induced NFκB signaling by EGFR activation in the colonic epithelium represents a novel mechanism through which EGFR restrains inflammatory responses. EGFR inhibits NFκB through a novel adapter protein, CCDC50, which functions independently of TNFAIP3, and current work is underway to determine this mechanism. This study uncovers a previously underappreciated protective mechanism for EGFR to inhibit innate immune responses in the colon by blunting the response of colon epithelial cells to microbial triggers, and thus limiting subsequent chemokine production and inflammation. Thus, EGFR and CCDC50 are promising targets to modulate mucosal inflammation in IBD.(Equation is included in full-text article.).