Pemetrexed has been widely used in patients with advanced non-small cell lung cancer (NSCLC). The clinical relevance of polymorphisms of folate pathway genes for pemetrexed metabolism have not been fully elucidated yet. The aim of this study was to evaluate the expression levels of circulating miR-22, miR-24, and miR-34a, possibly involved in folate pathway, in NSCLC patients treated with pemetrexed compared with healthy controls and to investigate their impact on patient clinical outcomes. A total of 22 consecutive patients with advanced NSCLC, treated with pemetrexed-based chemotherapy and 27 age and sex matched healthy controls were included in this preliminary analysis. miR-22, miR-24, and miR-34a targets were identified by TargetScan 6.2 algorithm, validating the involvement of these microRNAs in folate pathway. MicroRNAs were isolated from whole blood and extracted with miRNAeasy Mini Kit (Qiagen). miRNA profiling was performed using Real-Time PCR. SPSS 17 was used to data analysis. miR-22, miR-24, and miR-34a were found upregulated (P<0.05) in NSCLC patients versus healthy controls. Higher expression levels were recorded for miR-34a. Nevertheless, significantly higher miR-22 expression was observed in patients developing progressive disease (P=0.03). No significant associations with clinical outcome were recorded for miR-24 and miR-34a. Albeit preliminary, these data support the involvement of miR-22, miR-24, and miR-34a in advanced NSCLC. The correlation between high expression of miR-22 in whole blood and the lack of response in pemetrexed treated NSCLC patients indicates that miR-22 could represent a novel predictive biomarker for pemetrexed-based treatment.