Vancomycin (VCM)-induced nephrotoxicity impedes its treatment applications. Thus, it is important to clarify the relevant mechanism. This study investigated phosphoprotein changes attributable to the VCM nephrotoxicity mechanisms. Biochemical, pathological and phosphoproteomic analyses based on C57BL/6 mice were performed to explore the mechanisms.VCM-treated mice showed increased levels of blood urea nitrogen and creatinine, and signs of acute tubular necrotic lesions. Phosphoproteomic profiling identified 3025 differentially phosphorylated phosphopeptides between the model and control group. Gene Ontology enrichment analysis demonstrated that Molecular Function “oxidoreductase activity” and Cellular Component “peroxisome” were markedly enriched. KEGG pathway analysis identified an enrichment in peroxisome pathway and PPAR (peroxisome proliferator-activated receptor) signaling pathways. Parallel reaction monitoring analysis revealed a significant downregulation of CAT, SOD-1, AGPS, DHRS4, and EHHADH at phosphorylation level by VCM. Notably, the phosphorylation of ACO, AMACR, and SCPX was downregulated by VCM, which are the fatty acid β-oxidation-related proteins involved in PPAR signaling pathways. The phosphorylated PEX5 involved in peroxisome biogenesis was upregulated by VCM. Collectively, these findings indicated that VCM-induced nephrotoxicity is closely associated with peroxisome pathway and PPAR signaling pathways. The current study provides important insight into the mechanisms of VCM nephrotoxicity and will aid in the development of preventive and therapeutic strategies against this nephropathy.