Abstract: Cerebral small vessel disease (CSVD) and dementia are increasingly prevalent in an aging world population. A major risk factor for CSVD and cognitive impairment is diabetes, but the underlying mechanism remains unclear. Methylglyoxal (MGO), a by-product of glycolysis and a major precursor in the formation of advanced glycation endproducts (AGEs), is increased in diabetes and has been directly linked to microvascular complications in diabetes. Whether MGO is implicated in CSVD and the diabetes-associated risk of stroke and cognitive impairment is unknown. To investigate the effect of MGO on brain microcirculation, we used in vivo models in which we increased MGO in plasma through exogenous application or increased endogenous MGO formation. We furthermore investigated whether MGO can pass through the blood-brain barrier from plasma using a human induced pluripotent stem cell (hiPSC) model of the brain endothelium. We found that the endogenous formation, but not the exogenous supplementation, of MGO is of importance in the development of cerebral microvascular dysfunction and cognitive impairment in otherwise healthy subjects. We believe this is most likely due to the high reactivity of MGO and the poor passage of MGO through the brain endothelium. These findings reveal the potential of MGO formation as a therapeutic target in CSVD.