Most non-small cell lung cancer (NSCLC) tumors with activating mutations of the epidermal growth factor receptor (EGFR) are initially responsive to first-generation, reversible EGFR tyrosine kinase inhibitors (TKI) such as gefitinib, but they subsequently develop resistance to these drugs through either acquisition of an additional T790M mutation of EGFR or amplification of the proto-oncogene MET. We have now investigated the effects of combination treatment with thymidylate synthase (TS)-targeting drugs and the second-generation, irreversible EGFR-TKI BIBW2992 on the growth of NSCLC cells with the T790M mutation. The effects of BIBW2992 on EGFR signaling and TS expression in gefitinib-resistant NSCLC cells were examined by immunoblot analysis. The effects of BIBW2992 and the TS-targeting agents S-1 (or 5-fluorouracil) or pemetrexed on the growth of gefitinib-resistant NSCLC cells were examined both in vitro and in vivo. The combination of BIBW2992 with 5-fluorouracil or pemetrexed synergistically inhibited the proliferation of NSCLC cells with the T790M mutation in vitro, whereas an antagonistic interaction was apparent in this regard between gefitinib and either of these TS-targeting agents. BIBW2992 induced downregulation of TS in the gefitinib-resistant NSCLC cells, implicating depletion of TS in the enhanced antitumor effect of the combination therapy. The combination of BIBW2992 and either the oral fluoropyrimidine S-1 or pemetrexed also inhibited the growth of NSCLC xenografts with the T790M mutation to an extent greater than that apparent with either agent alone. The addition of TS-targeting drugs to BIBW2992 is a promising strategy to overcome EGFR-TKI resistance in NSCLC with the T790M mutation of EGFR.