Lung cancer is one of the most lethal tumors and, although standard chemotherapy produces clinical response, there has been little improvement in prognosis. Therefore, research effort has focused on target-specific agents, such as sorafenib, which blocks both the RAF/MEK/ERK signalling pathways and receptors involved in neovascularization and tumor progression, including VEGFR-2 and c-Kit. We investigated whether sorafenib would be synergistic with gemcitabine against NSCLC cell lines. Human lung cancer cells A549, CALU-1, CALU-6, H23 and HCC 827 were treated with sorafenib and gemcitabine, alone or in combination, and the cytotoxicity was assessed with CellTiter 96 Non-radioactive cell proliferation kit. Cell cycle and apoptosis were investigated with flow cytometry and fluorescence microscopy, respectively. Moreover, the effects of drugs on Akt (S473), c-Kit (Y823) and ERK (pTpY185/187) phosphorylation were studied with ELISA. Finally, quantitative PCR analysis was performed to assess whether sorafenib and gemcitabine modulated the expression of genes related to drug activity. Gemcitabine and sorafenib synergistically interacted on the inhibition of cell proliferation, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index. Sorafenib reduced c-Kit and ERK activation and gemcitabine inhibited Akt phosphorylation. Moreover quantitative PCR showed that sorafenib modulated the expression of targets related to gemcitabine activity, while gemcitabine induced the expression of RKIP. These data demonstrate that sorafenib and gemcitabine synergistically interact against NSCLC cells, through suppression of Akt, c-Kit and ERK phosphorylation, induction of apoptosis and modulation of dCK, RRM1, RRM2 and RKIP gene expression. The association between traditional cytotoxic agents with new target-specific agents, such as sorafenib, is a challenge for both clinical and preclinical future investigations in lung cancer treatments. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.