Perinatal hypoxic-ischemic brain injury is an important cause of neurological deficits still causing mortality and morbidity in the early period of life. As efficient clinical or pharmaceutical strategies to prevent or reduce the outcome of perinatal hypoxic-ischemic brain damage are limited, the development of new therapies is of utmost importance. To evolve innovative therapeutic concepts, elucidation of the mechanisms contributing to the neurological impairments upon hypoxic-ischemic brain injury is necessary. Therefore, we aimed for the identification of proteins that are affected by hypoxic-ischemic brain injury in neonatal rats. To assess changes in protein expression two days after induction of brain damage, a 2D-DIGE based proteome analysis was performed. Among the proteins altered after hypoxic-ischemic brain injury, Calcineurin A, Coronin-1A, as well as GFAP were identified, showing higher expression in lesioned hemispheres. Validation of the changes in Calcineurin A expression by Western Blot analysis demonstrated several truncated forms of this protein generated by limited proteolysis after hypoxia-ischemia. Further analysis revealed activation of calpain, which is involved in the limited proteolysis of Calcineurin. Active forms of Calcineurin are associated with the dephosphorylation of Darpp-32, an effect that was also demonstrated in lesioned hemispheres after perinatal brain injury.