Antifolates are important for chemotherapy in non-small cell lung cancer (NSCLC). They mainly rely on reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) to enter cells. PCFT is supposed to be the dominant transporter of the two in tumors, as it operates optimally at acidic pH and has limited transport activity at physiological pH, whereas RFC operates optimally at neutral pH. In this study, we found RFC showed a slightly pH-dependent uptake of antifolates, with similar affinity values at pH 7.4 and 6.5. PCFT showed a highly pH-dependent uptake of antifolates, with an optimum pH of 6.0 for pemetrexed and 5.5 for methotrexate. The Michaelis-Menten constant (Km ) value of PCFT for pemetrexed at pH 7.4 was more than 10 times higher than that at pH 6.5. Interestingly, we found that antifolate accumulations mediated by PCFT at acidic pH were significantly affected by the efflux transporter, breast cancer resistance protein (BCRP). The highest pemetrexed concentration was observed at pH 7.0-7.4 after a 60-minute accumulation in PCFT-expressing cells, which was further evidenced by the cytotoxicity of pemetrexed, with the IC50 value of pemetrexed at pH 7.4 being one-third of that at pH 6.5. In addition, the in vivo study indicated that increasing PCFT and RFC expression significantly enhanced the antitumor efficacy of pemetrexed despite the high expression of BCRP. These results suggest that both RFC and PCFT are important for antifolates accumulation in NSCLC, although there is an acidic microenvironment and high BCRP expression in tumors. SIGNIFICANCE STATEMENT: Evaluating the role of reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) on antifolates accumulation in non-small cell lung cancer (NSCLC) is necessary for new drug designs. By using cell models, we found both RFC and PCFT were important for antifolates accumulation in NSCLC. Breast cancer resistance protein (BCRP) significantly affected PCFT-mediated antifolates accumulation at acidic pH but not RFC-mediated pemetrexed accumulation at physiological pH. High expression of PCFT or RFC enhanced the cytotoxicity and antitumor effect of pemetrexed.