Abstract: Objective To clarify the gene expression profile of renal endothelial cell subpopulations in a calcium oxalate kidney stone rat model, providing reference for understanding the role of renal endothelial cells in kidney damage mediated by kidney stones. Methods Adult male SD rats were selected, and a calcium oxalate kidney stone model of rats was established using a 1% ethylene glycol (EG) and 1% ammonium chloride (AC) method (experimental group). Male rats fed under normal conditions were used as the control group. Von Kossa staining was used to detect calcium salt crystal deposition in rat kidney tissues. Single-cell nucleus transcriptome sequencing (snRNA-seq) was performed to identify renal endothelial cell subpopulations, and differential analysis of the transcriptomes of renal endothelial cell subpopulations in the experimental and control groups was conducted. Bioinformatics methods, including Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis, and Gene Set Enrichment Analysis (GSEA), were applied to analyze the differential gene sets. Results In the experimental group, distinct calcium salt deposition areas were observed in the kidney tissue, appearing black or dark brown, indicating successful construction of the calcium oxalate kidney stone model. Single-cell nucleus transcriptome sequencing identified a total of 2,116 differential genes between the experimental and control groups in renal endothelial cell subpopulations, with 706 genes upregulated and 1,410 genes downregulated in the experimental group. Notable genes, including LOC299282, S100a11, and LOC100360841, were upregulated, while LOC361914, Slc7a13, and Tcf7 were downregulated, with statistical significance. KEGG pathway analysis showed alterations in the PI3K/Akt signaling pathway, Rap1 signaling pathway, HIF-1 signaling pathway, ribosome, endocrine system, adherens junctions, and cellular senescence in the experimental group, with statistical significance. Conclusions This study confirms the impact of calcium oxalate stones on the transcriptome of renal endothelial cells and provides a new theoretical basis for understanding the gene expression profile changes in renal endothelial cell subpopulations in kidney damage caused by kidney stone formation.