MicroRNAs (miRNAs) are endogenous small (19-24 nt long) noncoding RNAs that regulate gene expression in a sequence specific manner. An increasing association between miRNA and cancer has been recently reported. Lung cancer is globally responsible for 1.4 million deaths annually and is the leading cause of cancer-related deaths in both women and men. In this study, we investigated the miR-21 expression in non-small cell lung cancer (NSCLC) to evaluate their value in prognosis of this tumor. Here, we assess miR-21 expression in NSCLC and its clinical significance including survival analysis. The expression of miR-21 in matched normal and tumor tissues of NSCLC was evaluated using a quantitative real-time RT-PCR. A Kaplan-Meier survival curve was generated following a logrank test. It was observed that miR-21 expression was up-regulated in NSCLC tissues compared with noncancerous lung tissues (mean ± SD: 6.7 ± 2.3 vs. 3.7 ± 1.5, P < 0.001). The up-regulation of miR-21 in NSCLC cancer tissues was also significantly correlated with aggressive clinicopathological features. We found that the patients with high miR-21 expression have a higher tumor grade (P = 0.027) and are in higher risk of lymph node metastasis (P = 0.021). Moreover, the results of Kaplan-Meier analyses showed that NSCLC patients with the high miR-21 expression tend to have shorter overall survival and progression free survival (P < 0.001). The multivariate analysis clearly indicated that the high miR-21 expression in biopsy samples may be considered as an independent prognostic factor in NSCLC for decreased survival (RR 3.88; 95%CI, 2.47-6.11). Our data indicate the potential of miR-21 as a novel prognostic biomarker for NSCLC. Large well-designed studies with diverse populations and functional evaluations are warranted to confirm and extend our findings.