Activated protein C (APC) is a protease with anticoagulant and cytoprotective activities. APC is neuroprotective in rodent models of stroke. But, an APC variant with reduced anticoagulant activity, 3K3A‐APC, compared to wild‐type APC shows greater neuroprotection with no risk for bleeding in stroke models. To determine whether 3K3A‐APC exhibits species‐dependent neuroprotection similar to that as seen with wild‐type APC, we studied murine and human recombinant 3K3A‐APC mutants which show approximately 80% reduced anticoagulant activity. Murine 3K3A‐APC (0.2 mg/kg i.v.) administered at 4 h after embolic stroke improved substantially functional outcome and reduced by 80% the infract volume 7 days after stroke. Human 3K3A‐APC was neuroprotective after embolic stroke in mice, but at significantly higher concentrations (i.e. 2 mg/kg i.v.). Species‐dependent neuroprotection, i.e. murine > human 3K3A‐APC, was confirmed in a mouse model of permanent middle cerebral artery occlusion. Human 3K3A‐APC had by fivefold greater cytoprotective activity than murine 3K3A‐APC in oxygen‐glucose deprivation model in human brain endothelial cells, whereas murine 3K3A‐APC was by 2.5‐fold more potent than human 3K3A‐APC in a mouse model of NMDA‐induced neuronal apoptosis. Thus, 3K3A‐APC exhibits species‐dependent neuroprotection which should be taken into account when designing human trials for ischemic stroke with APC mutants.