Endocardial endothelium, which lines the chambers of the heart, is distinct in its origin, structure, and function. However, though functionally very important, no studies at protein level have been conducted so far characterizing endocardial endothelium. In this study, we used endothelial cells from pig heart to investigate if endocardial endothelial cells are distinct at the proteome level. Using a high throughput liquid chromatography-tandem mass spectrometry for proteome profiling and expression, we identified sets of proteins that belong to specific biological processes and metabolic pathways in endocardial endothelial cells supporting its specific structural and functional roles.