In aerobic conditions, the heart preferentially oxidizes fatty acids. However, during metabolic stress glucose becomes the major energy source and enhanced glucose uptake has a protective effect on heart function and cardiomyocyte survival. Thus, abnormal regulation of glucose uptake may participate in the development of cardiac disease in diabetics. Ketone bodies are often elevated in poorly-controlled diabetics and are associated with increased cellular oxidative stress. Thus, we sought to determine the effect of the ketone body -hydroxybutyrate (OHB) on cardiac glucose uptake during metabolic stress. We used 2,4-dinitrophenol (DNP), an uncoupler of the mitochondrial oxidative chain, to mimic hypoxia in cardiomyocytes. Our data demonstrated that chronic exposure to OHB provoked a concentration-dependent decrease of DNP action, resulting in 56% inhibition of DNP-mediated glucose uptake at 5 mM OHB. This was paralleled by a diminution of DNP-mediated AMPK and p38 MAPK phosphorylation. Chronic exposure to OHB also increased reactive oxygen species (ROS) production by 1.9-fold compared to control cells. To further understand the role of ROS in OHB action, cardiomyocytes were incubated with H₂O₂. Our results demonstrated that this treatment diminished DNP-induced glucose uptake without altering activation of the AMPK/p38 MAPK signaling pathway. Incubation with the antioxidant N-acetylcysteine partially restored DNP-mediated glucose but not AMPK/p38 MAPK activation. In conclusion, these results suggest that ketone bodies, through inhibition of the AMPK/p38 MAPK signaling pathway and ROS overproduction, regulate DNP action and thus cardiac glucose uptake. Altered glucose uptake in hyperketonemic states during metabolic stress may participate in diabetic cardiomyopathy.