Heat shock protein 27 (Hsp27), a recently discovered member of the heat shock protein family, is markedly induced in the brain after cerebral ischemia and other injury states. In non-neuronal systems, Hsp27 has potent cell death-suppressing functions. However, the mechanism of Hsp27-mediated neuroprotection has not yet been elucidated. Using transgenic and viral overexpression of Hsp27, we investigated the molecular mechanism by which Hsp27 exerts its neuroprotective effect. Overexpression of Hsp27 conferred long-lasting tissue preservation and neurobehavioral recovery, as measured by infarct volume, sensorimotor function, and cognitive tasks up to 3 weeks following focal cerebral ischemia. Examination of signaling pathways critical to neuronal death demonstrated that Hsp27 overexpression led to the suppression of the MKK4/JNK kinase cascade. While Hsp27 overexpression did not suppress activation of an upstream regulatory kinase of the MKK/JNK cascade, ASK1, Hsp27 effectively inhibited ASK1 activity via a physical association through its N-terminal domain and the kinase domain of ASK1. The N-terminal region of Hsp27 was required for neuroprotective function against in vitro ischemia. Moreover, knockdown of ASK1 or inhibition of the ASK1/MKK4 cascade effectively inhibited cell death following neuronal ischemia. This underscores the importance of this kinase cascade in the progression of ischemic neuronal death. Inhibition of PI3K had no effect on Hsp27-mediated neuroprotection, suggesting that Hsp27 does not promote cell survival via activation of PI3K/Akt. Based on these findings, we conclude that overexpression of Hsp27 confers long-lasting neuroprotection against ischemic brain injury via a previously unexplored association and inhibition of ASK1 kinase signaling.