Epithelial-to-mesenchymal transition (EMT) is a fundamental biological process whereby epithelial cells lose their polarity and undergo a transition to a mesenchymal phenotype. When cancer cells invade adjacent tissues, they use a mechanism akin to EMT, and understanding the molecular mechanisms that drive this transition will facilitate studies into new targets for prevention of metastasis. Extracellular stimuli, such as growth factors, and their cytosolic effectors cooperate to promote EMT. In highly fibrotic cancers like lung cancer, it is thought that extracellular matrix molecules, including collagen, can initiate signals that promote EMT. Here, we present data showing that collagen I induces EMT in non-small cell lung cancer cell lines, which is prevented by blocking transforming growth factor (TGF)-beta3 signaling. In addition, we show that collagen I-induced EMT is prevented by inhibitors of phosphoinositide 3-kinase and extracellular signal-related kinase signaling, which promotes transcription of TGF-beta3 mRNA in these cells. Thus, our data are consistent with the hypothesis that collagen I induces EMT in lung cancer cells by activating autocrine TGF-beta3 signaling. Epidermal growth factor also seems to initiate EMT via a TGF-dependent mechanism.