Cocaine and amphetamine-regulated transcript (CART) is a neuropeptide that protects brains against ischemic injury in vivo and in vitro. By using small interference RNA against CART(CARTi), this study shows that CART knockdown by CARTi downregulated exogenous and endogenous CART mRNA and protein expression in vivo and in vitro. Consequently, CART knockdown exacerbated neuronal cell death induced by oxygen and glucose deprivation (OGD). It also showed that CART knockdown increased infarct size in a mouse middle cerebral artery occlusion model. CART's protective effects are most likely mediated through the ERK 1/2 pathway, since ERK 1/2 phosphorylation, not that of p38 or JNK is activated in CART-treated neurons after OGD. Furthermore, neuroprotection of CART is abolished by CART knockdown and by pretreatment with ERK antagonist PD98059 and U0126, but not with p38 or JNK antagonists SB203580 or SP600125. These results provide further evidence that CART is an endogenous neuroprotective peptide against cerebral ischemia and it does so through the MAPK/ERK signaling pathway. Therefore, CART may be developed into a therapeutic agent for stroke-related brain injury.