Glucose oxidation is a major contributor to myocardial energy production and its contribution is orchestrated by insulin. While insulin increases myocardial glucose uptake, it can also directly stimulate glucose oxidation, independent of increasing glucose uptake and glycolysis, through activating pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. However, how insulin directly stimulates mitochondrial PDH is not known. Here, we show that insulin-stimulated mitochondrial translocation of protein kinase B (Akt) plays a prerequisite role in transducing insulin signal to mitochondria to stimulate glucose oxidation. We found that inhibition of mitochondrial Akt completely abolishes insulin-stimulated glucose oxidation, independent of any change in glucose uptake and glycolysis. This was also associated with an increase in myocardial oxygen consumption due to increased reliance on fatty acid oxidation at the expense of glucose that led to a significant decrease in cardiac efficiency. We also revealed a novel role of mitochondrial protein kinase C-delta (PKC-δ) as a negative feedback loop to regulate the insulin stimulation of glucose oxidation. Inhibition of mitochondrial PKC-δ mimics the stimulatory effect of insulin on glucose oxidation via enhancing mitochondrial translocation of Akt. Although it can be translocation following insulin stimulation, inhibition of mitochondrial glycogen synthase kinase-3 beta (GSK-3b) did not affect insulin-stimulated glucose oxidation, suggesting it has a negligible role in mediating the direct stimulation of insulin on glucose oxidation. Thus, we identify insulin-stimulated mitochondrial translocation of Akt as a major transmitter of the insulin signal from to the mitochondrial to directly stimulate glucose oxidation in a glycolysis-independent manner.