Mechanistic contributions of anaerobic microbiota to airway disease are poorly understood due to inherent limitations of existing laboratory models. To address this knowledge gap, we developed a co-culture approach that maintains an oxygen-limited apical epithelial microenvironment while host cells are oxygenated basolaterally. Reduced oxygen culture did not alter the physiology or gene expression of Calu-3 cells but sustained anaerobe-epithelial interactions for 24h without affecting bacterial or host cell viability. Anaerobe challenge led to increased expression of inflammatory marker genes and compromised integrity of apical mucins, leading to our hypothesis that anaerobe-host interactions prime the airways for chronic infection. Indeed, anaerobe pre-treatment of Calu-3 cells led to an increase in Pseudomonas aeruginosa colonization. This model system offers new insight into anaerobe-host interactions in airway disease pathophysiology and motivates further study of the lung, gut, and oral cavity, where etiological roles of anaerobes have been proposed but specific pathogenic mechanisms remain unclear.