Chronic obstructive pulmonary disease is the third leading cause of death worldwide with tobacco smoke being the most common causative factor. Airway surface liquid (ASL) is a thin layer that lines the luminal side of airways which plays a major role in in innate defense facilitating the clearance of inhaled pathogens as well. SPLUNC1 is a secreted protein in the upper airway that regulates ASL height and has anti-microbial activity against respiratory pathogens. Exposure of cigarette smoke (CS) to airway epithelia is known to induce inflammation, mucus hypersecretion, and depletion of the ASL volume which together results in in bacterial infection and mucus stasis. However whether CS affects SPLUNC1 activity directly is unknown. We investigated the role of CS on SPLUNC1 activity using an in vitro CS exposure model, ASL height measurements, mass spectrometry and antimicrobial assays. After exposing recombinant SPLUNC1 to CS we observed a reduction in the ability of SPLUNC1 to regulate ASL height (P<0.05) (N=6). Overnight dialysis of SPLUNC1 did not reverse this effect (N=6). Furthermore antimicrobial activity against Gram Negative bacteria such as Haemophilus influenzae and Pseudomonas aeruginosa was attenuated following CS exposure (P<0.05) (N=3). Further, analysis of smoked SPLUNC1 using mass spectrometry revealed oxidative modifications including tri-oxidation and crotonaldehyde, to the cysteine residues at positions 180 and 224 (N=3). Some of these oxidative modifications were also observed in endogenous SPLUNC1 from cell culture secretions. The exact mode of action derivatizing these cysteines by cigarette components warrants further investigation. We conclude that cigarette smoke modifies SPLUNC1 resulting in its loss of ASL height regulation and attenuation of anti-microbial activity. This data expands on the severe impact of cigarette smoking on lung physiology through inactivating SPLUNC1 function. The identification of modified secreted proteins in the airway following smoke exposure may serves as biomarkers that can be used to assess the toxicity of cigarette compounds. Funded by the Tobacco Centers of Regulatory Science. Grant Number: 5P50HL120100