Background: Thymidylate synthase (TS) has a predictive role in pemetrexed treatment of mesothelioma; however, additional chemoresistance mechanisms are poorly understood. Here, we explored the role of the reduced-folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) in antifolate resistance in mesothelioma. Patients and methods: PCFT, RFC and TS RNA and PCFT protein levels were determined by quantitative RT-PCR of frozen tissues and immunohistochemistry of tissue-microarrays, respectively, in two cohorts of pemetrexed-treated patients. Data were analyzed by t-test, Fisher's/log-rank test and Cox proportional models. The contribution of PCFT expression and PCFT-promoter methylation to pemetrexed activity were evaluated in mesothelioma cells and spheroids, through 5-aza-20-deoxycytidinemediated demethylation and siRNA-knockdown. Results: Pemetrexed-treated patients with low PCFT had significantly lower rates of disease control, and shorter overall survival (OS), in both the test (N=73, 11.3 versus 20.1 months, P=0.01) and validation (N=51, 12.6 versus 30.3 months, P=0.02) cohorts. Multivariate analysis confirmed PCFT-independent prognostic role. Low-PCFT protein levels were also associated with shorter OS. Patients with both low-PCFT and high-TS levels had the worst prognosis (OS, 5.5 months), whereas associations were neither found for RFC nor in pemetrexed-untreated patients. PCFT silencing reduced pemetrexed sensitivity, whereas 5-aza-20- deoxycytidine overcame resistance. Conclusions: These findings identify for the first time PCFT as a novel mesothelioma prognostic biomarker, prompting prospective trials for its validation. Moreover, preclinical data suggest that targeting PCFT-promoter methylation might eradicate pemetrexed-resistant cells characterized by low-PCFT expression.