MicroRNAs (miRNAs) are small non-coding RNAs frequently dysregulated in human malignancies. In this study, we analyzed the global expression profile of miR-181 in non-small cell lung cancer (NSCLC), as its participation in some other types of cancer has been suggested by previous reports. We found that miR-181 was downregulated both in NSCLC tissues and cell lines. Overexpression of miR-181 evidently inhibited A549 cell proliferation, migration, and invasion and promotes cell apoptosis. Moreover, we also found miR-181 reduction was associated with increased Bcl-2 levels and miR-181 was further suggested to exert its pro-apoptotic function mainly through targeting Bcl-2 expression. Taken together, our study implicates important roles of miR-181 in lung cancer pathogenesis and implicates its potential application in cancer therapy.