Developing myocardium is more dependent on glycolysis than adult myocardium, yet the effects of selectively inhibiting glycolysis versus oxidative phosphorylation on embryonic heart function have not been well-characterized. Accordingly, we investigated how selective metabolic inhibition affects membrane voltage and intracellular Ca (Ca_i) transients in embryonic mouse hearts, including their susceptibility to arrhythmias. A total of 136 isolated embryonic mouse hearts were exposed to either 1) 2-deoxyglucose (2DG, 10 mM) or iodoacetate (IAA, 0.1 mM) with 10 mM pyruvate in place of glucose to selectively inhibit glycolysis, or 2) the mitochondrial uncoupler protonophore carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone (FCCP, 500 nM) with 10 mM glucose present to selectively inhibit oxidative phosphorylation. Using confocal imaging, we found that mitochondrial membrane potential monitored tetramethylrhodamine methyl ester (TMRM, 200 nM) remained stable with 2DG or IAA, but depolarized within 5 minutes after exposure to FCCP. IAA and FCCP decreased heart rate, inhibited Cai transient amplitude, shortened action potential duration at 80% repolarization (APD₈₀), and prolonged atrioventricular (AV) conduction time to similar extents. While 2DG decreased heart rate and Ca_i transient amplitude, it did not significantly affect APD₈₀ and AV conduction time. In addition, spontaneous arrhythmias occurred in 77/136 embryonic hearts (57%) after exposure to IAA (28/53) or FCCP (49/83). There were no significant differences in the types or incidence of arrhythmias induced by IAA and FCCP. These data support the idea that both glycolysis and oxidative phosphorylation play a critical metabolic role, comparable to that of oxidative phosphorylation, in regulating cardiac function in the embryonic mouse heart.