Background: Oxaliplatin-induced peripheral neuropathy (OIPN) poses a significant challenge for patients with colorectal tumor, often resulting in treatment interruption or discontinuation and subsequent treatment failure. Herein, a longitudinal untargeted metabolomic study to reveal the metabolomic profiles and biomarkers associated with the progression of OIPN.Methods: A prospective cohort of 129 colorectal cancer patients receiving oxaliplatin-based chemotherapy was stratified into four OIPN severity grades (Level 0-3). Serum samples underwent untargeted LC-MS/MS metabolomic analysis, detecting 521 metabolites. Multivariate statistical models and SHAP-guided random forest algorithms were employed to prioritize biomarkers. Machine learning validation included six classifiers assessed via ROC-AUC.Results: The cumulative dose of Oxaliplatin chemotherapy plays an important role in OIPN. At the same time, our findings implied that the occurrence of OIPN may be associated with the progression of the disease and the patients’ tumor markers (CEA, CA19-9, CA72-4), as well as immune response and inflammation (ANC, PLT), and metabolic and liver function abnormalities (GGT and UA) (P<0.05).Multivariate statistical analysis combined with SHAP-guided machine learning identified six biomarkers, including thiabendazole, 1-methylxanthine, imidazol-5-yl-pyruvate, 5-hydroxypentanoic acid, spermidine, and 4’-oxolividamine that consistently distinguished OIPN patients (Level 1-3) from non-OIPN controls (Level 0). Machine learning models, validated across six classifiers, demonstrated near-perfect discrimination for early-stage OIPN (AUC nearly 1). However, differentiation between intermediate OIPN grades (Level 1 vs 2, Level 1 vs 3, Level 2 vs 3) yielded lower predictive accuracy (AUC: 0.549–0.843), likely due to cohort size limitations and reliance on subjective sensory-based grading. Pathway enrichment analysis highlighted dysregulation in ABC transporters, central carbon metabolism in cancer, amino acid metabolism, and linoleic acid metabolism, suggesting potential roles in OIPN pathogenesis.Conclusions: These findings suggest that the selected biomarkers could serve as a foundation for the prediction and management of OIPN in colorectal cancer patients.