MicroRNAs (miRNAs) have been reported to be involved in many human cancers and tumor progression. The dysregulation of miR-449a is found in many types of malignancies and is associated with tumor growth, migration, and invasion. However, its expression and function in non-small cell lung cancer (NSCLC) still remains unclear. In our study, miR-449a was found to be downregulated in both NSCLC tissues and cell lines, and low miR-449a expression was obviously associated with tumor differentiation, TMN stage, and poor overall survival (OS). Moreover, we demonstrated that miR-449a could inhibit tumor proliferation, migration, and invasion in NSCLC. We also confirmed that HMGB1 was a direct target gene of miR-449a in NSCLC with dual-luciferase reporter assay, and upregulation of HMGB1 could reverse the miR-449a-induced suppression of growth, migration, and invasion in NSCLC cells. Last, we found that miR-449a suppressed tumor initiation and development through the NF-κB signaling pathway. These results indicate that miR-449a functions as a tumor suppressor in NSCLC by targeting the HMGB1-mediated NF-κB signaling pathway in NSCLC.