Our previous studies have demonstrated that the JNK signaling pathway plays an important role in ischemic brain injury and is mediated via glutamate receptor 6. Others studies have shown that N-methyl-d-aspartate (NMDA) receptor is involved in the neuroprotection of ischemic preconditioning. Here we examined whether ischemic preconditioning down-regulates activation of the mixed lineage kinase-JNK signaling pathway via NMDA receptor-mediated Akt1 activation. In our present results, ischemic preconditioning could not only inhibit activations of mixed lineage kinase 3, JNK1/2, and c-Jun but also enhanced activation of Akt1. In addition, both NMDA (an agonist of NMDA receptor) and preconditioning showed neuroprotective effects. In contrast, ketamine, an antagonist of NMDA receptor, prevented the above effects of preconditioning. Further studies indicated that LY294002, an inhibitor of phosphoinositide 3-kinase that is an upstream signaling protein of Akt1, could block neuroprotection of preconditioning, and KN62, an inhibitor of calmodulin-dependent protein kinase, also achieved the same effects as LY294002. Therefore, both phosphoinositide 3-kinase and calmodulin-dependent protein kinase are involved in the activation of Akt1 in ischemic tolerance. Taken together, our results indicate that preconditioning can inhibit activation of JNK signaling pathway via NMDA receptor-mediated Akt1 activation and induce neuroprotection in hippocampal CA1 region.