Abstract: Carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, is implicated in tumor progression and treatment resistance. However, its role in non-small cell lung cancer (NSCLC) remains unclear. This study examined CBR1 expression in NSCLC tissues and cell lines, using gene interference and pharmacological inhibition to assess its impact on stemness, chemosensitivity, and quiescence, and to explore underlying mechanisms. Our findings indicate that CBR1 expression is elevated in NSCLC tissues and cell lines, and further increases in the presence of cisplatin (CDDP). Gene interference reducing CBR1 expression significantly decreased the percentage of cluster of differentiation 133 (CD133) ‐ sitive cells and the expression of organic cation / carnitine transporter 4 (OCT4) and SRY (sex determining region Y) ‐ ox 2 (SOX2), while enhancing CDDP chemosensitivity. The CBR1 ‐ specific inhibitor PP ‐ Me markedly increased CDDP cytotoxicity and reduced stemness. Additionally, CBR1 inhibition via sh ‐ CBR1 or Hydroxy ‐ PP‐ Me (PP ‐ Me) disrupted NSCLC cell quiescence, as shown by a decrease in G0 phase cells and p27 expression, alongside an increase in cyclin D1 and pRb expression. Furthermore, SET domain containing 4 (SETD4), which mediates stemness, chemosensitivity, and quiescence in NSCLC cells, was downregulated by sh ‐ CBR1 or PP ‐ Me treatment. Overexpression of SETD4 counteracted the enhanced chemosensitivity resulting from CBR1 inhibition. In A549 xenografts, combined PP ‐ Me and CDDP therapy significantly inhibited tumor growth compared to either treatment alone. In conclusion, CBR1 inhibition enhances CDDP chemosensitivity by suppressing stemness and quiescence in NSCLC.