Aim: The aim of this study was to investigate whether Gs-Rbl relieves the CoCl2-induced apoptosis of hypoxic neonatal rat cardiomyocytes and in which the role of glucose transporter-4 (GLUT-4). Methods: Gs-Rb1 (0, 10, 50, 100, 200, 400, and 500 μmol/L), adenine 9-β-D-arabinofuranoside (ara A, 500 μmol/L; AMPK inhibitor) and wortmannin (0.5μmol/L; PI3K inhibitor) only in combination with 200 μmol/L Gs-Rbl were administered in hypoxic cardiomyocytes, which were induced by 500 μmol/L COCl2 for 12 h. Then, the apoptotic rate (AR), 2-[3H]-deoxy- D-glucose (2-[3H]-DG) uptake, and the expression of GLUT-4 (including in plasma membrane, PM), phospho-AMPKα(Thr172), AMPKα and Akt in cells were assayed.Results: Compared with simple hypoxia (0 μmol/L Gs-Rb1), Gs-Rb1 greater than 10μmol/L significantly decreased the apoptotic rate (P<0.01) and significantly increased 2-[3H]-DG uptake (P<0.01), GLUT-4 content in cells and PM (P<0.01), AMPK activity (P<0.01) and Akt (P<0.01) levels in a dose-dependent manner. AMPK activity was completely suppressed by ara-A, just as Akt was suppressed by wortmannin. The AR, glucose uptake and GLUT-4 levels in cells and PM were partly down-regulated by ara-A or wortmannin.Conclusion: Gs-Rb1 may protect neonatal rat cardiomyocytes from apoptosis induced by CoCl2. The anti-apoptotic effect of Gs-Rb1 may occur by improving glucose uptake, in which GLUT-4 translocation and expression played a key role. Both the AMPK and the P13K/Akt pathways may take part in the anti-hypoxic efficacy of Gs-Rb1.