Purpose: To investigate the role of microRNA-122-5p in the pathogenesis of non-small cell lung cancer (NSCLC) and its underlying mechanism. Methods: A total of 72 pairs of NSCLC tissues and paracancerous tissues were collected. The expression level of microRNA-122-5p in NSCLC tissues and paracancerous tissues were detected by qRT-PCR (quantitative real-time polymerase chain reaction). The relationship between microRNA-122-5p expression and the clinical prognosis of NSCLC patients was then analyzed. Bioinformatics prediction and luciferase activity assay were performed to validate the direct binding of microRNA-122-5p and p53. Cell cycle, proliferation, and apoptosis were detected after microRNA-122-5p knockdown in NSCLC cells. The regulatory effect of microRNA-122-5p on promoting NSCLC development was detected by Western blot. Results: MicroRNA-122-5p was more overexpressed in NSCLC tissues than in paracancerous tissues. MicroRNA-122-5p expression was negatively correlated with survival rate of NSCLC patients. Besides, microRNA-122-5p knockdown remarkably inhibited the proliferation and cell cycle advancement and increased apoptosis of NSCLC cells. Luciferase reporter gene assay and Western blot results indicated that microRNA-122-5p downregulated p53 and activated PI3K-AKT pathway, thereby promoting NSCLC development. Conclusion: MicroRNA-122-5p is overexpressed in NSCLC. Overexpression of microRNA-122-5p promotes NSCLC development by downregulating p53 and activating PI3K-AKT pathway.