Poloxamer 188 (P188), a multiblock copolymer surfactant, has been shown to protect against ischemic tissue injury of cardiac muscle, testes and skeletal muscle, but the mechanisms have not been fully understood. In this study, we explored whether P188 had a protective effect against cerebral ischemia/reperfusion injury and its underlying mechanisms. The in vivo results showed that P188 significantly reduced the infarct volume, ameliorated the brain edema and neurological symptoms 24 h after ischemia/reperfusion. In the long-term outcome study, P188 markedly alleviated brain atrophy and motor impairments and increased survival rate in 3 weeks of post stroke period. Additionally, P188 protected cultured hippucampal HT22 cells against oxygen-glucose deprivation and reoxygenation (OGD/R) injury. The ability in membrane sealing was assessed with two fluorescent membrane-impermeant dyes. The results showed that P188 treatment significantly reduced the PI-positive cells following ischemia/reperfusion injury and repaired the HT22 cell membrane rupture induced by Triton X-100. In addition, P188 inhibited ischemia/reperfusion-induced activation of matrix metalloproteinase (MMP)-9 and leakage of Evans blue. Therefore, the present study concludes that P188 can protect against cerebral ischemia/reperfusion injury, and the protection involves multi-mechanisms in addition to the membrane resealing.