miR-154 has been proven to act as a tumor suppressor in several types of tumors. However, its role in non-small cell lung cancer (NSCLC) remains unclear. Thus, the aim of this study was to investigate the effects of miR-154 on NSCLC tumorigenesis and development. Using real-time quantitative PCR (qRT-PCR), we analyzed expression of miR-154 at the transcriptional level in 40 NSCLC tumor tissues and matched adjacent normal tissues and the correlation with clinicopathological features of the patients. The miR-154 mimic was stably transfected into NSCLC A549 cells, and the effects of miR-154 on cancer cell proliferation, colony formation, cell cycle arrest, apoptosis, migration and invasion in vitro, and on the growth of in vivo xenografts were investigated. miR-154 expression levels were significantly downregulated in the NSCLC compared to the corresponding non-cancerous lung tissues (P<0.05), and decreased miR-154 expression was significantly associated with metastasis (P<0.001), larger tumor size (P<0.001) and advanced TNM stage (P<0.001). Furthermore, transfection of the miR-154 mimic into the NSCLC A549 cells was able to inhibit cell proliferation, colony formation, invasion and migration, and induce cell apoptosis and G0/G1 cell cycle arrest. Enforced expression of miR-154 also suppressed the growth of cancer cell xenografts in vivo. These findings indicate that miR-154 may become a potential target for miR-based therapy of NSCLC.