New Findings: • What is the central question of this study? The aim of this study was to examine quantitatively whether glucose transporter 4 deficiency leads to more severe alterations in cardiac electrical activity during cardiac stress. • What is the main finding and what is its importance? When compared with hearts from corresponding control littermates, the measured epicardial potentials from the surface of cardiac-selective glucose transporter 4-ablated mouse hearts during hypoxia showed the following differences: (i) significant decreases in the maximal downstroke of the potentials; (ii) increased activation time; and (iii) greater alterations in the activation sequence. Insulin resistance, which characterizes type 2 diabetes, is associated with reduced translocation of glucose transporter 4 (GLUT4) to the plasma membrane following insulin stimulation, and diabetic patients with insulin resistance show a higher incidence of ischaemia, arrhythmias and sudden cardiac death. The aim of this study was to examine whether GLUT4 deficiency leads to more severe alterations in cardiac electrical activity during cardiac stress due to hypoxia. To fulfil this aim, we compared cardiac electrical activity from cardiac-selective GLUT4-ablated (G4H-/-) mouse hearts and corresponding control (CTL) littermates. A custom-made cylindrical 'cage' electrode array measured potentials (Ves) from the epicardium of isolated, perfused mouse hearts. The normalized average of the maximal downstroke of Ves (|dVes/dtmin|na), which we previously introduced as an index of electrical activity in normal, ischaemic and hypoxic hearts, was used to assess the effects of GLUT4 deficiency on electrical activity. The |dVes/dtmin|na of G4H-/- and CTL hearts decreased by 75 and 47%, respectively (P < 0.05), 30 min after the onset of hypoxia. Administration of insulin attenuated decreases in values of |dVes/dtmin|na in G4H-/- hearts as well as in CTL hearts, during hypoxia. In general, however, G4H-/- hearts showed a severe alteration of the propagation sequence and a prolonged total activation time. Results of this study demonstrate that reduced glucose availability associated with insulin resistance and a reduction in GLUT4-mediated glucose transport impairs electrical activity during hypoxia, and may contribute to cardiac vulnerability to arrhythmias in diabetic patients. © 2013 The Physiological Society.