Carbon monoxide (CO) is an endogenous product of mammalian cells generated by heme-oxygenase, presenting anti-apoptotic properties in several tissues. The present work demonstrates the ability of small amounts of exogenous CO to prevent neuronal apoptosis induced by excitotoxicity and oxidative stress in mice primary culture of cerebellar granule cells. Additionally, our data show that endogenous CO is a heme-oxygenase product critical for its anti-apoptotic activity. Despite being neuroprotective, CO also induces reactive oxygen species generation in neurons. These two phenomena suggest that CO induces pre-conditioning (PC) to prevent cell death. The role of several PC mediators, namely soluble guanylyl cyclase, nitric oxide (NO) synthase, and ATP-dependent mitochondrial K channel (mitoK(ATP)) was addressed. Inhibition of soluble guanylyl cyclase or NO synthase activity, or closing of mitoK(ATP) abolishes the protective effect conferred by CO. In addition, CO treatment triggers cGMP and NO production in neurons. Opening of mitoK(ATP), which appears to be critical for CO prevention of apoptosis, might be a later event. We also demonstrated that reactive oxygen species generation and de novo protein synthesis are necessary for CO PC effect and neuroprotection. In conclusion, CO induces PC and prevents neuronal apoptosis, therefore constituting a novel and promising candidate for neuroprotective therapies.