High extracellular glucose concentration was reported to suppress intracellular Ca2⁢ clearing through altered sarcoplasmic reticulum (SR) function. In the present study, we attempted to elucidate the effects of pyruvate and fatty acid on SR function and reveal the mechanistic link with glucose-induced SR dysfunction. For this purpose, SR Ca2⁢-uptake rate was measured in digitonin-permeabilized H9c2 cardiomyocytes cultured in various conditions. Exposure of these cells to 5 mM pyruvate for 2 days induced a significant suppression of SR Ca2⁢-uptake, which was comparable to the effects of high glucose. These effects were accompanied with decreased glucose utilization. However, pyruvate could not further suppress SR Ca2⁢-uptake in cells cultured in high glucose condition. Enhanced entry of pyruvate into mitochondria by dichloroacetate, an activator of pyruvate dehydrogenase complex, also induced suppression of SR Ca2⁢-uptake, indicating that mitochondrial uptake of pyruvate is required in the SR dysfunction induced by pyruvate or glucose. On the other hand, augmentation of fatty acid supply by adding 0.2 to 0.8 mM oleic acid resulted in a dose-dependent suppression of SR Ca2⁢-uptake. However, these effects were attenuated in high glucose-cultured cells, with no significant changes by oleic acid concentrations lower than 0.4 mM. These results demonstrate that (1) increased pyruvate oxidation is the key mechanism in the SR dysfunction observed in high glucose-cultured cardiomyocytes; (2) exogenous fatty acid also suppresses SR Ca2⁢-uptake, presumably through a mechanism shared by glucose.