Background <p>This study investigates HDAC3 as a potential immunotherapy biomarker in advanced non-small cell lung cancer (aNSCLC), focusing on its association with treatment response to immune checkpoint inhibitors (ICIs).</p> Methods <p>We employed a multi-phase approach in 78 aNSCLC patients with 138 plasma samples, starting with a discovery phase that identified differential autoantibodies (AAbs) using proteomic analysis in responders and non-responders. In the validation phase, we assessed AAb levels at multiple time points. Additionally, immunohistochemistry and multiple immunofluorescence (<i>n</i> = 21) were used to validate HDAC3 expression in FFPE samples, single-cell RNA sequencing (<i>n</i> = 26) was performed to explore gene expression differences, cell and animal experiments were performed.</p> Results <p>We identified 127 differential AAbs, with five key AAbs (HDAC3, METTL21C, HSPB3, SPACA7, and SPPL2B) consistently linked to prognosis pre- and post-treatment (<i>p</i> &lt; 0.05). A risk score model based on these AAbs effectively predicted progression-free survival. Furthermore, HDAC3 expression correlated with significant pathway enrichments and was associated with higher TGFβ1, PD-L1 infiltration and lower CD8⁺ T cells infiltration (<i>p</i> &lt; 0.05). <i>HDAC3</i> knockdown significantly inhibited cell proliferation, impaired colony formation, and induced G0/G1 phase arrest in lung cancer cells. Preclinical models demonstrated that RGFP966, an HDAC3 inhibitor, combined with anti–PD-1 therapy enhanced CD8⁺ T cell infiltration (<i>p</i> &lt; 0.05).</p> Conclusion <p>Our findings underscore HDAC3’s role as a biomarker for predicting ICI response in aNSCLC and suggest its potential as a therapeutic target, paving the way for future studies on HDAC3-targeted therapies to improve immunotherapy outcomes.</p> Clinical trial number <p>not applicable.</p>