In a number of large clinical studies, concurrent administration of the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) with cytotoxic chemotherapy has failed to improve the survival rate in unselected patients with advanced non-small cell lung cancer (NSCLC). The purpose of the current study was to investigate the antitumor effects of gefitinib in combination with docetaxel in EGFR-TKI-sensitive, primary resistant and acquired resistant human lung cancer cell lines and the associated molecular mechanisms. EGFR-TKI-sensitive and EGFR-TKI-resistant human lung cancer cell lines were exposed to gefitinib or docetaxel alone, or in combination. Cell viability was assessed using the MTT assay. Cell cycle distribution and apoptosis were measured by flow cytometry and alterations in signaling pathways were examined by immunoblotting. The cytotoxic interaction between docetaxel and gefitinib was determined by combination index (CI) analysis. Coadministration of gefitinib and docetaxel was observed to result in superior inhibition of tumor cell proliferation, however, increased rates of apoptosis were only observed in EGFR-TKI-sensitive cells, whereas, antagonistic activity was observed in the EGFR-TKI-resistant cell lines. Gefitinib arrested the cell cycle at the G1 phase, whereas docetaxel arrested the cell cycle at the S phase. In addition, in cells exhibiting a synergistic interaction between gefitinib and docetaxel, an increase in p-EGFR and p-AKT was observed following chemotherapy exposure. By contrast, in cells exhibiting no change or a decrease in p-EGFR and p-AKT following docetaxel treatment, an antagonistic interaction between the two agents was observed. In conclusion, the combination of docetaxel and gefitinib generated synergistic effects in EGFR-TKI-sensitive cells and antagonistic effects in EGFR-TKI-primary and acquired resistant cells, suggesting that EGFR-TKIs, combined with docetaxel, may be beneficial to NSCLC patients with EGFR mutations. The results also indicate that the interactions between gefitinib and docetaxel may be associated with the effect of docetaxel on EGFR phosphorylation.