The proximal tubule (PT) is a nephron segment that is responsible for the majority of solute and water reabsorption in the kidney. Each of its sub-segments have specialized functions; however, little is known about the genes and proteins that determine the oxidative phosphorylation capacity of the PT sub-segments. This information is critical to understanding kidney function and will provide a comprehensive landscape of renal cell adaptations to injury, physiologic stressors, and development. This study analyzed three immortalized murine renal cell lines (PT S1, S2, and S3 segments) for protein content and compared them to a murine fibroblast cell line. All three proximal tubule cell lines generate ATP predominantly by oxidative phosphorylation while the fibroblast cell line is glycolytic. The proteomic data demonstrates that the most significant difference in proteomic signatures between the cell lines are proteins known to be localized in the nucleus followed by mitochondrial proteins. Mitochondrial metabolic substrate utilization assays were performed using the proximal tubule cell lines to determine substrate utilization kinetics thereby providing a physiologic context to the proteomic dataset. This data will allow researchers to study differences in nephron-specific cell lines, between epithelial and fibroblast cells, and between actively respiring cells and glycolytic cells. SIGNIFICANCE: Proteomic analysis of proteins expressed in immortalized murine renal proximal tubule cells was compared to a murine fibroblast cell line proteome. The proximal tubule segment specific cell lines: S1, S2 and S3 are all grown under conditions whereby the cells generate ATP by oxidative phosphorylation while the fibroblast cell line utilizes anaerobic glycolysis for ATP generation. The proteomic studies allow for the following queries: 1) comparisons between the proximal tubule segment specific cell lines, 2) comparisons between polarized epithelia and fibroblasts, 3) comparison between cells employing oxidative phosphorylation versus anaerobic glycolysis and 4) comparisons between cells grown on clear versus opaque membrane supports. The data finds major differences in nuclear protein expression and mitochondrial proteins. This proteomic data set will be an important baseline dataset for investigators who need immortalized renal proximal tubule epithelial cells for their research.