Background: CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden; however, the biological mechanisms of CC16 on pulmonary epithelial responses remain undefined. Methods: Mouse tracheal epithelial cells (MTECS) from CC16 sufficient (WT) and CC16 deficient (CC16-/-) mice were grown at an air-liquid interface and mass spectrometry and quantitative proteomics used to investigate the impact of CC16 on apically secreted proteins. Results: Eight antimicrobials were significantly repressed from CC16-/- MTECs, 6 of which were validated in and human nasal epithelial cells (HNECs). Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) was the most differentially expressed antimicrobial protein (66-fold) and was the focus of this study. We discovered that CC16 enhances epithelial-driven SPLUNC1 production via signaling through the receptor complex Very Late Antigen-2 (VLA-2) and that rCC16 enhances pulmonary SPLUNC1 production in vivo. Conclusions: Our findings identify VLA-2 as a new receptor for CC16 and a novel role of CC16 in the airway epithelium by up-regulating antimicrobials.?Clinical Implications: These findings could impact host responses to respiratory pathogens for individuals in which low CC16 levels have been described, including in Asthma, COPD and in Cystic Fibrosis patients.