Diabetes mellitus (DM) is a major metabolic disorder currently affecting over 380 million people worldwide. DM can induce oxidative stress in the body leading to the generation of elevated levels of the endogenous reactive carbonyl species (RCSs), including methylyglyoxal (MGO) which can elicit vascular dysfunction. Elevated MGO is believed to reduce the pool of endothelial progenitor cells (EPCs) responsible for maintaining the vasculature. This study isolated EPCs from the blood and characterizing them, measured endogenous level of MGO in blood and tissues during DM and investigated the mechanism(s) whereby MGO can elicit a reduction of EPCs or cell death. Young adult male Sprague Dawley rats weighing 180-200g were rendered diabetic using a single dose of intraperitoneal injection (ip) containing45 mg/kg body weight streptozotocin (STZ) dissolved in citrate buffer. Age-matched animals were given the same volume (0.3 ml of the buffer alone. Fasting blood glucose was assessed four days prior to STZ injection and on the day (8 weeks later) when the animals were humanely killed for experimentation using an ACCU-CHEK Aviva glucose meter (Roche, Mannheim, Germany) to confirm DM. EPCs were incubated with MGO for 24 hours using varying concentrations (0-500 μM) to determine the effect of MGO on EPC viability using MTT assay.Western blot analysis was used to determine the relative levels of the MGO scavenging enzyme glyoxalase following its overexpression. Confocal imaging was used to assess the effect of MGO on cytoplasmic and mitochondrial calcium and ROS production. STZ-treated rats had significantly (Mean ± SEM; Student’s t-test; p<0.01) higher blood glucose values and reduced heart to body mass ratio relative to controls. Blood glucose levels were 452±7.8 mg/dl and 116.7±4.38 mg/dl and heart to body mass ratios (g/100g body weight) were 0.28±0.08 and 0.3±0.07 for diabetic (n=5) compared to control (n=5) rats. Serum insulin (ng/ml) was 1.07±0.16 and 0.29±0.05 in diabetic and control rats, respectively.The results show that MGO level is significantly ( p<0.05) elevated in serum, whole blood, heart and brain tissues, but not in the kidneys, during DM compared to healthy age-matched controls. In contrast, the number of EPCs decreased significantly (p<0.05) in the blood of diabetic rats compared to control. The MTT assay showed that increasing concentrations of MGO had the ability to kill EPCs with an EC50 of 100 µM. Over-expression of glyoxylase enzyme blunted the effect of MGO on cell viability. Confocal microscopic results have indicated significant (p<0.05) time-dependent increases in both cytosolic Ca2+ and mitochondrial Ca2+ release from either fluo-3 or rhodamine-loaded EPCs treated with 100 µM MGO, respectively. MGO can also elevate mitochondrial ROS production from EPCs. It is concluded that the RCS-generated MGO has the ability to induce death of EPCs through a mechanism involving superoxide production and mitochondrial calcium overloading.