Diabetes causes hyperglycemia, which can create a stressful environment for cardiac microvascular endothelial cells (CMECs). To investigate the impact of diabetes on the cellular metabolism of CMECs, we assessed glycolysis by quantifying the extracellular acidification rate (ECAR), and mitochondrial oxidative phosphorylation (OXPHOS) by measuring cellular oxygen consumption rate (OCR), in isolated CMECs from wild-type (WT) hearts and diabetic hearts (db/db) using an extracellular flux analyzer. Diabetic CMECs exhibited a higher level of intracellular reactive oxygen species (ROS), and significantly reduced glycolytic reserve and non-glycolytic acidification, as compared to WT CMECs. In addition, OCR assay showed that diabetic CMECs had increased maximal respiration, and significantly reduced non-mitochondrial oxygen consumption and proton leak. Quantitative PCR (qPCR) showed no difference in copy number of mitochondrial DNA (mtDNA) between diabetic and WT CMECs. In addition, gene expression profiling analysis showed an overall decrease in the expression of essential genes related to β-oxidation (Sirt1, Acox1, Acox3, Hadha, and Hadhb), tricarboxylic acid cycle (TCA) (Idh-3a and Ogdh), and electron transport chain (ETC) (Sdhd and Uqcrq) in diabetic CMECs compared to WT CMECs. Western blot confirmed that the protein expression of Hadha, Acox1, and Uqcrq was decreased in diabetic CMECs. Although lectin staining demonstrated no significant difference in capillary density between the hearts of WT mice and db/db mice, diabetic CMECs showed a lower percentage of cell proliferation by Ki67 staining, and a higher percentage of cellular apoptosis by TUNEL staining, compared with WT CMECs. In conclusion, excessive ROS caused by hyperglycemia is associated with impaired glycolysis and mitochondrial function in diabetic CMECs, which in turn may reduce proliferation and promote CMEC apoptosis.