Background: Chronic obstructive pulmonary disease (COPD) is a risk factor for non-small cell lung cancer (NSCLC), and cigarette smoking is the main risk factor for both. The pathogenesis of tumorigenesis remains unclear, making novel biomarkers necessary. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and contribute to both COPD and NSCLC pathogeneses. However, whether each COPD and NSCLC have a specific miRNA signature is unclear. Aims: We profiled the miRNA lung expression in NSCLC and COPD patients, and healthy controls (HCs). Methods: Formalin-fixed and paraffin-embedded (FFPE) lung sections were collected from HCs, NSCLC, and COPD patients to extract miRNA. miRNA associated with NSCLC and COPD pathogenesis were quantified by RT-PCR. miRNA target genes were identified by in silico analysis. Results: hsa-miR-33a-5p and 320a-3p were significantly increased in NSCLC patients vs. HCs and COPD patients. In contrast, 34a-5p was significantly increased in COPD patients vs. HCs and NSCLS patients. Interestingly, both hsa-miR- 33a-5p and 320a-3p, the levels of which were the highest in NSCLC, limit cell proliferation by upregulating genes involved in nuclear factor kappa B (NF-kB)-mediated cell apoptosis. In contrast, the hsa-miR-34a-5p, which was the highest in COPD, promotes tumorigenesis by positively regulating genes associated with both neurogenic locus notch homolog protein 1 (NOTCH1) and B-cell lymphoma 2 (Bcl2) signaling. Conclusions: NSCLC and COPD are characterized by miRNA with opposite roles, with inhibition and promotion of cell proliferation, respectively. Thus, miRNA could serve as potential biomarkers for disease progression and response to treatment.