S100B is a glial‐derived protein that is a well‐established biomarker for severity of neurological injury and prognosis for recovery. Cell‐based and clinical studies have implicated S100B in the initiation and maintenance of a pathological, glial‐mediated proinflammatory state in the central nervous system. However, the relationship between S100B levels and susceptibility to neurological injury in vivo has not been determined. We used S100B transgenic (Tg) and knockout (KO) mice to test the hypothesis that overexpression of S100B increases vulnerability to cerebral hypoxic‐ischemic injury and that this response correlates with an increase in neuroinflammation from activated glia. Postnatal day 8 Tg mice subjected to hypoxia‐ischemia showed a significant increase in mortality compared with KO and wild‐type mice. Tg mice also exhibited greater cerebral injury and volume loss in the ischemic hemisphere after an 8‐day recovery, as assessed by histopathology and magnetic resonance imaging. Measurement of glial fibrillary acidic protein and S100B levels showed a significant increase in the Tg mice, consistent with heightened glial activation and neuroinflammation in response to injury. This is the first demonstration to our knowledge that overexpression of S100B in vivo enhances pathological response to injury. Ann Neurol 2004;56:61–67