Hypersecretion of mucus characterizes many inflammatory airway diseases, including asthma, chronic bronchitis, and cystic fibrosis. Excess mucus causes airway obstruction, reduces pulmonary function, and can lead to increased morbid-ity and mortality. MicroRNAs are small non-coding pieces of RNA which regulate other genes by binding to a com-plementary sequence in the target mRNA. The microRNA miR-21 is upregulated in many inflammatory conditions and, interestingly, miR-21 has been shown to target the mRNA of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS), a protein that is an important regulator of airway mucin (the solid component of mucus) secretion. In these studies, we determined that exposure of primary, well-differentiated, normal human bronchial epithelial (NHBE) cells to the pro-inflammatory stimulus lipopolysaccharide (LPS) increased expression of both miR-21 and MARCKS in a time-dependent manner. To investigate whether miR-21 regulation of MARCKS played a role in mucin secretion, two separate airway epithelial cell lines, HBE1 (papilloma virus transformed) and NCI-H292 (mucodepidermoid derived) were utilized, since manipulation of miR-21 is performed via transfection of commercially-available miR-21 inhibitors and mimics/activators. Treatment of HBE1 cells with LPS caused concentration-dependent increases in expression of both miR-21 and MARCKS mRNA and protein. The miR-21 inhibitor effectively reduced levels of miR-21 in the cells, coincident with an increase in MARCKS mRNA expression over time as well as enhanced mucin secretion, while the miR-21 mimic/activator increased levels of miR-21, which coincided with a decrease in expression of MARCKS and a decrease in mucin secretion. These results suggest that miR-21 is increased in airway epithelial cells following exposure to LPS, and that miR-21 downregulates expression of MARCKS, which may decrease mucin secretion by the cells. Thus, miR-21 may act as a negative feedback regulator of mucin secretion in airway epithelial cells, and may do so, at least in part, by downregulating expression of MARCKS.