Vascular endothelial cells show considerable diversity, particularly when located within a specific organ such as the heart [1]. Myocardial capillaries, lined by cardiac microvascular endothelial cells (CMECs), are critical in the development of end-organ damage [2], and show adaptations in view of their close association with cardiomyocytes, whose function they tightly regulate [3]. Little is known about the total protein expression patterns in CMECs. The aim of the study was to perform a comprehensive large-scale proteomic analysis of adult rat CMECs under normoxic, baseline and hypoxic conditions in order to gain more information on their functional properties. Methods: Adult rat (Rattus norvegicus) CMECs were purchased and passaged until generation 4-7. CMECs were incubated under normoxia (21% O2), or hypoxia (<1% O2) for 24h. Samples were then subjected to SDS-PAGE/in-gel trypsinisation or filter-aided sample preparation (FASP) before separation of peptides by nano-LC chromatography and analysis by a LTQ Orbitrap Velos mass spectrometer. Proteins were identified with 4 different protein database search engines. Finally, protein lists were analysed for functional and pathway annotations.Results: A total of 1388 proteins were identified. The majority of proteins were expressed in the cytosol (28.3% of the proteome; p=1.1 x 10-63), followed by mitochondrial (22.4%; p=1.1 x 10-27), cytoskeletal (15.4%; p=3.5 x 10-15), and vesicular (11.5%; p=2.3 x 10-14) proteins. The top 5 biological processes represented in the CMEC proteome were: protein localisation (9.8%; p=1.5 x 10-10), oxidation reduction (9.6%; p=2.7 x 10-11), protein transport (8.8%; p=4.9 x 10-11), translation (8.4%; p=2.4 x 10-14), and vesicle-mediated transport (7.7%;p=2.1 x 10-11). The hypoxia protocol induced demonstrable injury (1.5-fold and 2.2-fold increase in necrosis and apoptosis respectively; p<0.05). 267 new proteins were expressed, whereas 32 proteins were lost in hypoxia. Five out of the 10 upregulated proteins in hypoxia were glycolytic enzymes. The cytosolic protein fraction increased to 47% of the hypoxic CMEC proteome, whereas mitochondrial proteins decreased to 16%. There was a significant presence of ribonucleoproteins (26.3%; p= 7.8 x 10-23), and translation (24.5%) and glycolysis (6%) were more prominent vs normoxia.Apoptotic and hypoxia response proteins were overexpressed in hypoxia (9% and 4.8% respectively).Conclusions: This is the first comprehensive characterisation of the CMEC proteome under normoxic and hypoxic conditions. Normoxic CMECs demonstrated significant mitochondrial protein expression, which decreased after hypoxia. Conversely, glycolytic proteins increased in hypoxia suggesting a metabolic switch in the cells. Vesicular and protein transport proteins were prominently expressed in the CMECs, indicative of the capillary-derived origin of the cells.