We have previously reported that glutamate acting on NMDA receptors attenuated IGF-1 pro-survival signaling and its protective effect in cultured neurons. In this study, we investigate whether IGF-1 signaling was suppressed by glutamate in vivo, and whether this effect of glutamate was implicated in ischemic brain damage. Our results showed that exogenous glutamate injected by i.c.v. decreased phosphorylation of IGF-1 receptors and Akt, and this effect of glutamate was reversed by NMDA antagonist MK-801 but not by non-NMDA antagonist DNQX. NMDA exhibited similar effects of glutamate. Endogenous glutamate, which was induced by focal cerebral ischemia, gradually reduced the phosphorylation of IGF-1 receptors and Akt in a time-dependent manner. Moreover, IGF-1 injected by i.c.v. failed to stimulate phosphorylation of IGF-1 receptors and Akt after 180. min MCAO, and the protective effect was abolished. Pre-treatment of MK-801 restored the phosphorylation of IGF-1 receptors and Akt by IGF-1. In parallel, IGF-1 successfully rescued infarct area after 180. min MCAO. These findings suggest that glutamate interferes with IGF-1 signaling in vivo by activating NMDA receptors, and thereby shorten the therapeutic window of IGF-1 against focal cerebral ischemia. © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society.