Transforming growth factor beta1 (TGF-beta1) is a cardinal cytokine in the pathogenesis of airway remodeling, and promotes epithelial-to-mesenchymal transition (EMT). As a molecular interaction between TGF-beta1 and Jun N-terminal kinase (JNK) has been demonstrated, the goal of this study was to elucidate whether JNK plays a role in TGF-beta1-induced EMT. Primary cultures of mouse tracheal epithelial cells (MTEC) from wild-type, JNK1-/- or JNK2-/- mice were comparatively evaluated for their ability to undergo EMT in response to TGF-beta1. Wild-type MTEC exposed to TGF-beta1 demonstrated a prominent induction of mesenchymal mediators and a loss of epithelial markers, in conjunction with a loss of trans-epithelial resistance (TER). Significantly, TGF-beta1-mediated EMT was markedly blunted in epithelial cells lacking JNK1, while JNK2-/- MTEC underwent EMT in response to TGF-beta1 in a similar way to wild-type cells. Although Smad2/3 phosphorylation and nuclear localization of Smad4 were similar in JNK1-/- MTEC in response to TGF-beta1, Smad DNA-binding activity was diminished. Gene expression profiling demonstrated a global suppression of TGF-beta1-modulated genes, including regulators of EMT in JNK1-/- MTEC, in comparison with wild-type cells. In aggregate, these results illuminate the novel role of airway epithelial-dependent JNK1 activation in EMT.