Human MUC1 (Muc1 in animals) is an extensively O-glycosylated membrane-tethered mucin expressed on the surface of epithelial cells and some cells of the hematopoietic system. Recently, we showed that the hamster Muc1 on Chinese hamster ovary (CHO) cells served as a binding site for Pseudomonas aeruginosa (PA) through interaction between bacterial flagellin and the Muc1 ectodomain. Because CHO cells are known to produce an atypical pattern of protein glycosylation, we determined whether or not PA interacted with MUC1 endogenously expressed on human airway epithelial cells. Knock down of MUC1 expression in bronchial (NuLi-1) or alveolar (A549) epithelial cells by RNA interference significantly reduced PA binding to the cells. Conversely, over-expression of MUC1 in HEK293 cells increased bacterial adherence. By confocal microscopy, PA and MUC1 were colocalized on the surface of NuLi-1 cells. Taken together, these results confirm our previous observations in CHO cells and suggest that MUC1 serves as a binding site for PA on the surface of airway epithelial cells, which may have important consequences in the pathogenesis of PA lung infections.