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This study performs untargeted metabolomics on mouse serum and quantitative proteomics on xenograft tumors to explore the potential anti-tumor mechanisms of Inonotus obliquus polysaccharides (IOPs) against non-small cell lung cancer (NSCLC), revealing candidate pathways that warrant further experimental validation. Building on our prior discovery of IOP-induced apoptosis, we combined functional assays with multi-omics analyses using a well-characterized IOPs preparation (average molecular weight: 4.5 × 10⁴ Da; polysaccharide content: 70.3%; primarily composed of glucose). IOPs potently inhibited proliferation (MTT/colony formation) in LLC and H520 cells and suppressed tumor growth in an allograft model. Untargeted metabolomics of mouse serum revealed IOP-driven downregulation of lipid metabolism. Quantitative proteomics of tumors showed that IOPs regulate nuclear processes (transcription, DNA repair) and modulate the tumor immune microenvironment, as evidenced by enriched pathways of neutrophil extracellular trap (NET) formation, cell adhesion molecules, and Th1/Th2 cell differentiation. Collectively, these findings propose a potential working model in which IOPs may exert anti-tumor effects through coordinated nuclear regulation, metabolic reprogramming, and immune modulation. Our results provide preliminary evidence that IOPs may act beyond direct cytotoxicity, potentially involving systemic regulatory pathways. Further studies are needed to validate these findings and to assess its therapeutic potential in NSCLC.