Abstract: Objective: This study aimed to investigate the therapeutic potential and direct molecular targets of dioscin, a natural steroid saponin, in the progression of renal cell carcinoma (RCC). Methods: The anti-tumor phenotypes of dioscin were evaluated in vitro using Cell Counting Kit-8 (CCK-8), transwell and flow cytometry assays. Biotinylated dioscin probes coupled with mass spectrometry, molecular docking, and site-directed mutagenesis were employed to identify and validate direct intracellular targets. Mitochondrial function was assessed via Seahorse OCR analysis, ROS quantification and JC-1 staining. Subcutaneous xenograft mouse models were established to verify the in vivo efficacy of dioscin. Results: Dioscin profoundly suppressed RCC cell proliferation and metastasis while inducing dose-dependent apoptosis. Mechanistically, dioscin directly bound to the T62, T63 and T76 residues of FABP5, an oncogenic lipid chaperone upregulated in RCC tissues. This targeted engagement obstructed FABP5-mediated fatty acid transport, provoking intracellular triglyceride depletion, blunted mitochondrial respiration, massive ROS accumulation, and the collapse of mitochondrial membrane potential. The systemic administration of dioscin robustly retarded tumor growth in vivo, while showing no overt hepatotoxicity, nephrotoxicity or major organ damage. Conclusions: Our findings unveiled the existence of a novel dioscin/FABP5/mitochondrial homeostasis signaling axis and demonstrated that dioscin may represent a promising and selective therapeutic candidate for restraining RCC progression.