The chemokine CX3CL1 and its receptor CX3CR1 are constitutively expressed in the nervous system. In this study we investigated the effect of CX3CL1 in an in vivo model of permanent middle cerebral artery occlusion (pMCAO) in rats and mice and report that exogenous CX3CL1 reduced ischemia-induced cerebral infarct size, neurological deficits and caspase-3 activation. CX3CL1-induced neuroprotective effects were long-lasting, being observed up to fifty days after pMCAO in rats. The neuroprotective action of CX3CL1 in different models of brain injuries is mediated by its inhibitory activity on microglia and, in vitro, requires the activation of adenosine receptor 1 (A1R). We show that, in the presence of the A1R antagonist DPCPX and in A1R-/- mice, the neuroprotective effect of CX3CL1 upon pMCAO was abolished, indicating the critical importance of the adenosine system in CX3CL1 protection also in vivo. In apparent contrast with the above reported data but in agreement with earlier findings, cx3cr1GFP/GFP and cx3cl1-/- mice had less severe brain injury upon pMCAO and the administration of exogenous CX3CL1 increased brain damage in cx3cl1-/- ischemic mice. We also report that CX3CL1 induced a different phagocytic activity in wt and cx3cl1-/- microglia, in vitro, upon co-treatment with the medium conditioned by neurons damaged by OGD. All together, these data suggest that acute administration of CX3CL1 reduces ischemic damage via an adenosine-dependent mechanism and that the absence of constitutive CX3CL1-CX3CR1 signaling changes the outcome of microglia-mediated effects upon CX3CL1 administration to ischemic brain.