Heat shock protein 27 (HSP27) exerts cytoprotection against many cellular insults including cerebral ischemia. We previously showed that intravenous injection of HSP27 purified from human lymphocytes (hHSP27) significantly reduced infarct volume following cerebral ischemia-reperfusion injury, while recombinant HSP27 (rHSP27) was less effective. Phosphorylation is important for HSP27 function, and hHSP27 was more highly phosphorylated than rHSP27. We hypothesized that MAPKAP kinase 2 in vitro-phosphorylated rHSP27 (prHSP27) might increase its brain protection. Mice underwent transient 1-h MCAO, and then received tail-vein injections of one of the following 1 h after reperfusion: hHSP27 as positive control, rHSP27, prHSP27, or bovine serum albumin (BSA) as control. We measured infarct volume, neurological deficits, neurological severity, physiological parameters, cell-death, oxidative stress, and inflammatory response. Compared with BSA controls (34.4±2.8mm³, n=5), infarct volume was reduced by 69% in the hHSP27 positive-control group (10.5±4.6mm³, P<0.001, n=5), 19% following rHSP27 (27.8±4.2mm³>, P<0.05, n=5), and 49% following prHSP27 (17.5±4.5mm³, P<0.001, n=9). Compared to the rHSP27 and BSA-treated groups, prHSP27 also reduced functional deficits, and significantly suppressed apoptosis, oxidative stress, and inflammatory responses. Here, we showed the superior neuroprotective effects of phosphorylated HSP27 by administering prHSP27. prHSP27 may be a useful therapeutic agent to protect against acute cerebral ischemic stroke.