<p>The survival rate of patients diagnosed with non-small cell lung cancer (NSCLC) are significantly influenced by drug resistance and metastasis with cancer stem cells (CSCs). MicroRNAs (miRNAs) impact on the progression of various human diseases, including different types of cancers. This research seeks to clarify how miR-34a-5p influences CSC characteristics and resistance to cisplatin (DDP) in NSCLC. Initially, the levels of expression for miR-34a-5p, KRT5, and DSP were analyzed in both NSCLC tissue samples and cell lines through RT-qPCR and western blot techniques. Subsequently, sphere formation assays, CCK-8 assays, and flow cytometry tests were carried out to evaluate CSCs and their resistance to DDP in NSCLC. The expression of genes related to stemness, such as SOX2, CD133, and ALDH, was assessed using western blotting methods. Functional experiments were also conducted to investigate the regulatory mechanisms linked to miR-34a-5p, KRT5, and DSP. Finally, the effect of miR-34a-5p on the CSC phenotype and resistance to DDP was validated using a xenograft mouse model. Results showed that, in NSCLC patients, miR-34a-5p was found to be decreased, while KRT5 and DSP expressions were elevated. By targeting the KRT5/DSP axis, miR-34a-5p was shown to suppress CSC properties and enhance DDP sensitivity in NSCLC cells. miR-34a-5p directly bound to and inhibited KRT5, resulting in the downregulation of DSP expression. Furthermore, the overexpression of miR-34a-5p in mice resulted in reduced tumor growth. Collectively, this study elucidates the regulatory role of miR-34a-5p on CSC properties, providing insights that may aid in overcoming drug resistance in NSCLC.</p>