Background-Limb remote ischemic preconditioning (RIPC) protects against brain injury induced by stroke, but the underlying protective mechanisms remain unknown. As hypoxia inducible factor 1 alpha (HIF-1 alpha) is neuroprotective in stroke and mediates neuroinflammation, we tested the hypothesis that HIF-1 alpha is a key factor of RIPC against stroke by mediating inflammation. Methods and Results-Stroke was induced by transient middle cerebral artery occlusion in rats, and RIPC was conducted in both hind limbs. The HIF-1 alpha mRNA was examined by quantitative reverse transcription polymerase chain reaction after RIPC. In addition, inflammatory cytokines in the peripheral blood and brain were measured using the AimPlex multiplex immunoassays. Data showed that RIPC reduced the infarct size, improved neurological functions, and increased HIF-1 alpha mRNA levels, interleukin (IL)-4, and IL-10 protein levels in the peripheral blood. Intraperitoneal injection of the HIF activator, dimethyloxaloylglycine, reduced the infarct size and inhibited interferon-c protein levels, while promoting IL-4 and IL-10 protein levels, while decreasing interferon-c protein levels in both the peripheral blood and ischemic brain. In addition, injection of dimethyloxaloylglycine had a synergistic effect with RIPC on reducing infarction and improving neurological functions, as well as decreasing interferon-c in the peripheral blood and ischemic brain. In contrast, injection of the HIF inhibitor, acriflavine hydrochloride, abolished the protective effects of RIPC on infarction, and reduced IL-4 and IL-10 protein levels in both the peripheral blood and ischemic brain. Conclusions-We conclude that HIF-1 alpha plays a key role in RIPC, likely mediated by a systemic modulation of the inflammatory response.