Intracerebral hemorrhage (ICH) is a devastating form of stroke. Misoprostol, a synthetic prostaglandin E₁ (PGE₁) analog and PGE₂ receptor agonist, has shown protection against cerebral ischemia. In this study, we tested the efficacy of misoprostol in the 12-month-old mice subjected to 1 of 2 complementary ICH models, the collagenase model (primary study) and blood model (secondary study, performed in an independent laboratory). We also investigated its potential mechanism of action. Misoprostol posttreatment decreased brain lesion volume, edema, and brain atrophy and improved long-term functional outcomes. In the collagenase-induced ICH model, misoprostol decreased cellular inflammatory response; attenuated oxidative brain damage and gelatinolytic activity; and decreased high-mobility group box 1 (HMGB1) expression, Src kinase activity, and interleukin-1β expression without affecting cyclooxygenase-2 expression. Furthermore, HMGB1 inhibition with glycyrrhizin decreased Src kinase activity, gelatinolytic activity, neuronal death, and brain lesion volume. Src kinase inhibition with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) decreased gelatinolytic activity and brain edema and improved neurologic function but did not decrease HMGB1 protein level. These results indicate that misoprostol protects brain against ICH injury through mechanisms that may involve the HMGB1, Src kinase, and matrix metalloproteinase-2/9 pathways.