To elucidate the mechanisms underlying the development of pemetrexed resistance in NSCLC, we established two pemetrexed-resistant sublines in two lung cancer cell lines, one carrying an EGFR mutation and the other retaining wild-type EGFR. In the present study, we demonstrated for the first time that the upregulation of the FGF2-FGFR1 pathway could confer pemetrexed resistance in lung cancer cells regardless of EGFR status. Here, FGF2 is linked to lung cancer.