Sorafenib is a multi-targeted agent and has been reported to have potent antitumor effects against various types of tumors, including human non-small cell lung cancer (NSCLC). In this study, we explored in vitro the antitumor effects of sorafenib alone and in combination with gemcitabine in epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant human lung cancer cell lines and the related molecular mechanisms. The NSCLC cell lines A549 (mutant KRAS), H1666 (mutant BRAF) and H1975 (mutant EGFR-T790M) were treated with sorafenib and gemcitabine alone and in combination. The cytotoxicity was assessed by MTT assay, cell cycle distribution was analyzed by flow cytometry, and alterations in signaling pathways were analyzed by western blotting. We found that sorafenib exhibited dose-dependent growth inhibition in all three EGFR-TKI-resistant NSCLC cell lines. When sorafenib was combined with gemcitabine, synergistic activity was observed in the A549 and H1666 cells and antagonistic activity was observed in the H1975 cells. Sorafenib arrested the cell cycle at the G1 phase, whereas gemcitabine arrested the cell cycle at the S phase. Sorafenib inhibited C-RAF and p-ERK in the A549 cells and B-RAF and p-ERK in the H1666 and H1975 cells. The molecular mechanism of this synergism is that RAF/MEK/ERK which are activated by gemcitabine are efficiently suppressed by simultaneously administered sorafenib. By contrast, the mechanism of antagonism may be due to mutual interference with the cell cycle in the H1975 cells. In conclusion, we found that sorafenib exhibits antiproliferative effects in EGFR-TKI-resistant NSCLC cell lines and when combined with gemcitabine demonstrates synergistic activity in A549 and H1666 cells but antagonistic activity in H1975 cells.