An acquired mutation (T790M) in the epidermal growth factor receptor (EGFR) accounts for half of all relapses in non-small cell lung cancer (NSCLC) patients who initially respond to EGFR kinase inhibitors. In this study, we demonstrated for the first time that EGFR-T790M interacts with the cytoskeletal components, myosin heavy chain 9 (MYH9) and β-actin, in the nucleus of H1975 cells carrying the T790M-mutant EGFR. The interactions of EGFR with MYH9 and β-actin were reduced in the presence of blebbistatin, a specific inhibitor for the MYH9-β-actin interaction, suggesting that the EGFR interaction with MYH9 and β-actin is affected by the integrity of the cytoskeleton. These physical interactions among MYH9, β-actin, and EGFR were also impaired by CL-387,785, a kinase inhibitor for EGFR-T790M. Furthermore, CL-387,785 and blebbistatin interacted in a synergistic fashion to suppress cell proliferation and induce apoptosis in H1975 cells. The combination of CL-387,785 and blebbistatin enhanced the down-regulation of cyclooxygenase-2 (COX-2), a transcriptional target of nuclear EGFR. Overall, our findings demonstrate that disrupting EGFR interactions with the cytoskeletal components enhanced the anti-cancer effects of CL-387,785 against H1975 cells, suggesting a novel therapeutic approach for NSCLC cells that express the drug-resistant EGFR-T790M.