Increasing matrix stiffness caused by the extracellular matrix (ECM) deposition surrounding cancer cells is accompanied by epithelial-mesenchymal transition (EMT). Here, we show that expression levels of EMT marker genes along with discoidin domain receptor 2 (DDR2) can increase upon matrix stiffening. DDR2 silencing by short hairpin RNA downregulated EMT markers. Promoter analysis and chromatin immunoprecipitation revealed that c-Myb and LEF1 may be responsible for DDR2 induction during cell culture on a stiff matrix. Mechanistically, c-Myb acetylation by p300, which is upregulated on the stiff matrix, seems to be necessary for the c-Myb-and-LEF1-mediated DDR2 expression. Finally, we found that the c-Myb-DDR2 axis is crucial for lung cancer cell line proliferation and expression of EMT marker genes in a stiff environment. Thus, our results suggest that DDR2 regulation by p300 expression and/or c-Myb acetylation upon matrix stiffening may be necessary for regulation of EMT and invasiveness of lung cancer cells.