Here we report that 5'-monophosphate (AMP)-activated protein kinase (AMPK) agonist A-769662 inhibited hydrogen peroxide (H2O2)-induced viability loss and apoptosis of human and mouse osteoblast cells. H2O2-induced moderate AMPK activation in osteoblast cells, which was enhanced by A-769662. Inactivation of AMPK by its inhibitor compound C, or by target shRNA-mediated silencing and kinase dead (KD) mutation exacerbated H2O2-induced cytotoxicity in osteoblast cells. A-769662-mediated protective effect against H2O2 was also blocked by AMPK inhibition or depletion. A-769662 inhibited reactive oxygen species (ROS) accumulation by H2O2 in osteoblast cells. Meanwhile, H2O2-induced ATP depletion was inhibited by A-769662, but was aggravated by compound C. Further, H2O2 induced AMPK-dependent and pro-survival autophagy in cultured osteoblast cells, which was enhanced by A-769662. Our results suggested that activation of AMPK by H2O2 is anti-apoptosis and pro-survival in osteoblast cells, probably due to its anti-oxidant, pro-autophagy and ATP preservation abilities, and A-769662-mediated cell-protective effect in osteoblast cells requires AMPK activation. Our study suggests that A-769662 might be further investigated as a novel anti-osteonecrosis agent.