Deeper mechanistic understanding of lung adenocarcinoma (non–small cell lung carcinoma, or NSCLC), a leading cause of cancer-related deaths overall, may lead to more effective therapeutic strategies. In analyzing NSCLC clinical specimens and cell lines, we discovered a uniform decrease in miR-186 (MIR186) expression in comparison with normal lung tissue or epithelial cell lines. miR-186 expression correlated with patient survival, with median overall survival time of 63.0 or 21.5 months in cases exhibiting high or low levels of miR-186, respectively. Enforced overexpression of miR-186 in NSCLC cells inhibited proliferation by inducing G₁–S checkpoint arrest. Conversely, RNA interference–mediated silencing miR-186 expression promoted cell-cycle progression and accelerated the proliferation of NSCLC cells. Cyclin D1 (CCND1), cyclin-dependent kinase (CDK)2, and CDK6 were each directly targeted for inhibition by miR-186 and restoring their expression reversed miR-186–mediated inhibition of cell-cycle progression. The inverse relationship between expression of miR-186 and its targets was confirmed in NSCLC tumor xenografts and clinical specimens. Taken together, our findings established a tumor-suppressive role for miR-186 in the progression of NSCLC. Cancer Res; 73(2); 756–66. ©2012 AACR.