Abnormal lipid metabolism is an independent risk factor for kidney injury, significantly altering the associated gene expression, particularly in single kidney models. This study investigates the impact of high-fat diet-induced lipid metabolism on podocyte injury in uninephrectomized mice. Using targeted lipidomics analysis and podocyte-specific assays, the modification of lipid profiles attributed to a high-fat diet and the development of podocyte injury caused by lipid metabolism in mice that underwent unilateral nephrectomy were examined. Mice that underwent unilateral nephrectomy and were fed with a high-fat diet for 13 weeks exhibited progressive renal dysfunction, including the accumulation of lipid droplets in podocytes, vacuolization of tubular cells, and glomerular hypertrophy. Liquid chromatography-triple quadrupole mass spectrometry confirmed a significant increase in cholesteryl ester 20:4 levels in the podocytes of these mice. In vitro, cholesteryl ester 20:4 treatment reduced mitochondrial respiration capacity and mitochondrial glycolysis in podocytes. Furthermore, the treatment led to alterations in the protein expression levels associated with lipid metabolism and transport, mitochondrial activity, and autophagy, including ATP binding cassette subfamily A member 1 (ABCA1), carnitine palmitoyltransferase 1 A (CPT1A), acyl-CoA cholesterol acyltransferase (ACAT), nuclear respiratory factor ½ (NRF½), dynamin-1-like protein (DRP1), and p62. Transcriptome sequencing analysis revealed impaired gene expression, which was associated with the progression of renal fibrosis in unilateral nephrectomy mice with a high-fat diet. Specifically, the expression of matrix metalloproteinases and collagen genes, including fibronectin and collagen IV, was upregulated, indicating fibrosis progression. In conclusion, lipidomics analysis identifies cholesteryl ester 20:4 as a key lipid metabolite accumulating in podocytes, which is associated with mitochondrial dysfunction and abnormal autophagy. This accumulation potentially contributes to structural and functional deterioration in the kidney and highlights its role in kidney damage and its potential as a therapeutic target in metabolic kidney diseases.