Rationale: Induced mainly by cigarette smoking, chronic obstructive pulmonary disease (COPD) is a global public health problem characterized by progressive difficulty in breathing and increased mucin production. Cigarette smoke is enriched in acrolein, a reactive irritant that can induce excessive mucin production in laboratory animals. In nonsmoking subjects with COPD, we previously found that acrolein was generated at concentrations that could activate matrix metalloproteinase 9 (MMP9). In this study, we examine whether acrolein alters expression and activation of MMP14, a critical membrane-bound endopeptidase that can initiate a MMP activation cascade. Measurements and Main Results: In a cell-free system, sub-micromolar acrolein concentrations equivalent to those found in COPD sputum directly activated MMP14 protein. An acrolein adduct was identified on cysteine 319 in the hemopexin-like domain of MMP14. In cells, MMP14 activity also is thought to be regulated by post-translational cleavage of prepro-MMP14 by proprotein convertase and membrane insertion. In human airway epithelial cells, acrolein stimulated MMP14 protein activity and this was inhibited by a proprotein convertase inhibitor, hexa-D-arginine. In the airway epithelium of COPD subjects, immunoreactive MMP14 protein is increased. In mouse lung, acrolein or tobacco smoke increased immunoreactive MMP14 protein and acrolein increased MMP14 activity. This led us to postulate that acrolein also could increase MMP14 mRNA levels. In human airway epithelial cells, acrolein increased MMP14 transcripts, which could be inhibited by an epidermal growth factor receptor (EGFR) neutralizing antibody (LA1), an EGFR kinase inhibitor (AG1478), or metalloproteinase inhibitor (GM6001). Acrolein-induced MMP14 transcripts were diminished by mitogen activated protein kinase 3/2 (MAPK3/2) (PD98059) or MAPK8 (SP600125) inhibitors, but not a MAPK14 (ML3403) inhibitor. In human airways epithelial cells, decreasing the MMP14 protein and activity in vitro by small interfering (si)RNA to MMP14 diminished the acrolein-induced mucin (MUC5AC) transcript increase. In acrolein-exposed mice or doxycycline-regulatable transgenic mice with lung-specific transforming growth factor-alpha (an EGFR ligand) expression, lung MMP14 and MUC5AC levels increased and these effects were diminished in mice treated with an EGFR kinase inhibitor, erlotinib. Conclusions: Together, these findings implicate acrolein-induced MMP14 expression and activity in mucin production in COPD.