Brain ischemic tolerance is an endogenous neuroprotective mechanism, whereby an experience of non-lethal ischemic episode (preconditioning; PC) produces resilience to subsequent lethal ischemia. We previously showed that PC caused activation of astrocytes and a subsequent upregulation of P2X7 receptors (P2X7Rs), activation of which induced ischemic tolerance. However, the downstream signals of P2X7Rs responsible for the ischemic tolerance remain unknown. Here we show that astrocytic P2X7R-dependent lactate release has an indispensable role for this event. Using a middle cerebral artery occlusion model, we found that extracellular lactate levels during lethal ischemia were significantly increased in mice that experienced PC, and the increase depended on P2X7Rs. In the in vitro experiments, although stimulation of astrocytes with P2X7R agonist BzATP had no effect on the protein levels of MCT1 and MCT4, which were responsible for lactate efflux in astrocytes, BzATP induced the plasma membrane translocation of these MCTs via their chaperone CD147. Importantly, CD147 was increased in activated astrocytes after PC, and CD147 blocking antibody abolished astrocyte-mediated lactate release and ischemic tolerance. Taken together, our findings suggest that astrocytes induce ischemic tolerance by P2X7R-dependent lactate release.