Stimulation of glucose uptake in response to ischemic metabolic stress is important for cardiomyocyte function and survival. Chronic exposure of cardiomyocytes to fatty acids (FA) impairs the stimulation of glucose uptake, whereas induction of lipid droplets (LD) is associated with preserved glucose uptake. However, the mechanisms by which LD induction prevents glucose uptake impairment remain elusive. We induced LD with either tetradecanoyl phorbol acetate (TPA) or 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Triacylglycerol biosynthesis enzymes were inhibited in cardiomyocytes exposed to FA ± LD inducers, either upstream (glycerol-3-phosphate acyltransferases; GPAT) or downstream (diacylglycerol acyltransferases; DGAT) of the diacylglycerol step. Although both inhibitions reduced LD formation in cardiomyocytes treated with FA and LD inducers, only DGAT inhibition impaired metabolic stress-stimulated glucose uptake. DGAT inhibition in FA plus TPA-treated cardiomyocytes reduced triacylglycerol but not diacylglycerol content, thus increasing the diacylglycerol/triacylglycerol ratio. In cardiomyocytes exposed to FA alone, GPAT inhibition reduced diacylglycerol but not triacylglycerol, thus decreasing the diacylglycerol/triacylglycerol ratio, prevented PKCδ activation and improved metabolic stress-stimulated glucose uptake. Changes in AMP-activated Protein Kinase activity failed to explain variations in metabolic stress-stimulated glucose uptake. Thus, LD formation regulates metabolic stress-stimulated glucose uptake in a manner best reflected by the diacylglycerol/triacylglycerol ratio.