AbstractEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKI) are strongly recommended for non‐small‐cell lung cancer (NSCLC) patients harbouring active EGFR mutations, while drug resistance makes exploring resistance mechanisms and seeking effective therapeutic strategies urgent endeavours. Thymidylate synthetase (TYMS or TS) is a dominant enzyme in thymidylate nucleotide metabolism. In this study, we found a positive correlation between TS expression and overall survival (OS) and disease‐free survival (DFS) in lung adenocarcinoma. The examination of gene sets from 140 NSCLC patients received EGFR‐TKI therapy demonstrated a negative correlation between high TS expression and the efficacy of EGFR‐TKI therapy. 24 tissue specimens from NSCLC patients exhibited upregulated TS mRNA expression in NSCLC patients resistant to gefitinib. The NSCLC cell PC9 and HCC827 sensitive to gefitinib and relatively resistant PC9/GR and HCC827/GR cells were used to demonstrate the knockdown of TS restored the sensitivity of resistant cells to gefitinib. Furthermore, pemetrexed effectively suppressed TS‐mediated thymidylate metabolism and induced ROS generation, DNA damage and cellular senescence, thereby hampering cancer progression and restoring sensitivity to gefitinib. Our findings illuminate the potential mechanism of TS‐triggered gefitinib resistance and indicate inhibition of TS by pemetrexed can potentiate the effect of gefitinib in NSCLC. Pemetrexed combined with gefitinib has potent anti‐progression potential in gefitinib‐resistant NSCLC. This study suggests that NSCLC patients with both high TS expression and EGFR‐driving mutations might benefit more from a combination strategy of EGFR‐TKI and pemetrexed‐based chemotherapy than EGFR‐TKI monotherapy, which has profound clinical implications and therapeutic value.