Epithelial to mesenchymal transition 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 epithelial to mesenchymal transition, 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, together with their cytosolic effectors cooperate to promote epithelial to mesenchymal transition. In highly fibrotic cancers like lung cancer, it is thought that extracellular matrix molecules including collagen can also initiate signals that promote epithelial to mesenchymal transition. Here, we present data showing that collagen I induces epithelial to mesenchymal transition in non-small cell lung cancer cell lines, which is completely prevented by blocking transforming growth factor-beta 3 signaling. In addition, we show that collagen I -induced EMT is prevented by inhibitors of phophoinositide 3-kinase and extracellular signal related kinase signaling, which promotes transcription of transforming growth factor-beta 3 mRNA in these cells. Thus, our data are consistent with the hypothesis that collagen I induces epithelial to mesenchymal transition in lung cancer cells by activating autocrine transforming growth factor-beta 3 signaling. Interestingly, epidermal growth factor also appears to initiate EMT via a transforming growth factor dependent mechanism.