Recently, accumulating evidence indicates that dysregulation of miRNAs is associated with the initiation and progression of cancer. MiR-101 has been reported down-regulated in various types of cancer. The aim of this study was to investigate the expression profile of miR-101 and its target gene Mcl-1 in NSCLC and to assess their clinical significance. QRT-PCR was used in the detection of miR-101 and Mcl-1 mRNA expression both in NSCLC tissue and in adjacent normal lung tissue. Immunohistochemistry and Western blot analysis were used in the detection of Mcl-1 protein expression. The clinicopathological implications of these molecules were analyzed statistically. Survival analysis was performed to assess prognostic significance. Down-regulation of miR-101 was associated with overexpression of Mcl-1 mRNA in NSCLC tissue when compared with corresponding normal tissue, with a negative correlation (r = -0.724, P < 0.01). MiR-101 expression was significantly associated with pathological stage (P = 0.004) and lymph node involvement (P = 0.012). Overexpression of Mcl-1 was associated with pathological grade (P = 0.022) and lymph node involvement (P = 0.017). A comparison of survival curves of low versus high expressers of miR-101 and Mcl-1 revealed a highly significant difference in NSCLC (P < 0.05), which suggests that reduced expression of miR-101 versus overexpression of Mcl-1 is associated with a poorer prognosis. Our results suggest that down-regulation of miR-101 may result in enhanced expression of Mcl-1 in NSCLC, which consequently favored tumor progression. MiR-101 and Mcl-1 may play important roles as biomarkers for prognosis and therapeutic targets in NSCLC.