Objective <p>This study explored the mechanism of RUNX1/NSUN2/FABP5 promoting immunotherapy resistance in non-small cell lung cancer (NSCLC).</p> Methods <p>NSCLC patients received anti-programmed cell death protein 1 (PD-1) therapy. In vitro A549 and NCI-H520 cells and in vivo xenograft mouse models were established. RUNX1, NSUN2, FABP5, PD-L1, and lipid metabolic profiles were analyzed using RT-qPCR, Western blot, and biochemical assays. Gain- and loss-of-function experiments were performed to assess the roles of RUNX1, FABP5, and NSUN2 in NSCLC. CD8⁺ T cells in tumor tissues were assessed through flow cytometry. A NSCLC cell-CD8⁺ T cell co-culture system was established, with T cell activation and effector cytokine secretion determined. The mechanism of the RUNX1/NSUN2/FABP5 axis in NSCLC was elucidated by dual-luciferase, ChIP, MeRIP, RIP, and Actinomycin D assays.</p> Results <p>RUNX1 was highly expressed in immunotherapy-resistant NSCLC. RUNX1 was positively correlated with elevated serum lipids and negatively associated with CD8⁺ T cell activation in tumor tissues. RUNX1 promoted PD-L1 expression and resistance to anti-PD-1 therapy in vivo via FABP5-mediated lipid metabolic reprogramming. RUNX1 regulated FABP5-mediated lipid metabolic reprogramming, enhanced PD-L1 expression, and induced CD8⁺ T cell dysfunction. FABP5 knockdown inhibited lipid metabolism and partially reversed the effects of RUNX1 in NSCLC. RUNX1 promoted NSUN2 transcription, which in turn increased m⁵C methylation of FABP5 and enhanced FABP5 mRNA stability. NSUN2 knockdown partially averted RUNX1’s effect in NSCLC.</p> Conclusion <p>RUNX1 alters m⁵C methylation levels by regulating NSUN2 transcription to promote FABP5-mediated lipid metabolic reprogramming, which in turn enhances PD-L1 expression and consequently induces immune resistance in NSCLC.</p>