Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/ MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. Weshow that, in the male mouse brain, progesterone is mainly metabolized via 5 alpha-reduction leading to 5 alpha-dihydroprogesterone (5 alpha-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3 alpha,5 alpha-tetrahydroprogesterone (3 alpha,5 alpha-THP). In the female mouse brain, levels of 5 alpha-DHP and 3 alpha,5 alpha-THP are lower and levels of 20 alpha-DHP are higher than in males. After MCAO, levels of progesterone and 5 alpha-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5 alpha-DHPcontribute to the resistance of neural cells to ischemic damage, weinactivatedPRselectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes.