The protease, activated protein C (APC) and its variants provide pharmacological benefits in many murine preclinical injury models, including neuroprotection for ischemic stroke and mortality reduction for sepsis. The in vivo mechanism of action for APC in ischemic stroke and sepsis injury models, similar to extensive in vitro studies using cultured cells, involves protease activated receptor (PAR) 1-mediated biased signaling. Cell culture studies showed that APC/PAR1 signaling requires β-Arrestin 2, an intracellular scaffold protein, and that β-Arrestin 2-dependent, APC-initiated signaling can distinctly alter a wide variety of intracellular signaling pathways. It is unclear whether in vitro studies can be extrapolated to in vivo mechanisms of action. This study employed a proximal transient Middle Cerebral Artery Occlusion model to study the neuroprotective actions of the signaling-selective APC variant, 3K3A-APC, in β-arrestin 2 deficient (Arrb2-/-) mice. Based on the size and shape of brain injury areas, 3K3A-APC significantly limited brain injury in control mice to a relatively small, localized area whereas these protective effects were lost in Arrb2-/- mice. Thus, these data indicate that the major in vitro mechanism of action that requires β-arrestin 2 for APC/PAR1 biased signaling is central to the in vivo mechanism of action for APC’s neuroprotection.