Subcortical white matter stroke constitutes up to 30% of all stroke subtypes, occurs initially as silent infarcts in the white matter (WM), and contributes significantly to the development of vascular dementia. Activated protein C (APC) is a plasma serine protease that is capable of antithrombotic, anti-inflammatory, anti-apoptotic, and cell-signaling activities. 3K3A-APC, a recombinant variant of APC with reduced (>90%) anticoagulant activity, engineered to reduce APC-associated bleeding risk while retaining normal cell-signaling activity, has shown benefits in preclinical models of ischemic stroke with big infarcts involving large brain arteries and gray matter. A multicenter Phase 2 randomized clinical trial has enrolled 110 patients to assess the safety, pharmacokinetics, and efficacy of human recombinant 3K3A-APC in acute ischemic stroke patients following endovascular thrombolysis (NCT02222714). In this study, subcortical WM stroke model was induced by vasoconstrictor N5-(1-iminoethyl)-L-ornithine, dihydrochloride (L-Nio). L-Nio (Calbiochem) was injected into subcortical WM of the anterior cingulum (AC) of the corticolimbic circuit or the subcortical white matter ventral to the mouse forelimb motor cortex using the Neurostar motorized ultra-precise small animal stereotaxic instrument (Model 963SD). Mice received 0.2 mg/kg intravenously of recombinant murine 3K3A-APC or saline at 4, 24, 48 and 72 h after stroke. The results indicate that 3K3A-APC significantly reduces the lesion volume and protected blood brain barrier damage 3 days after stroke, and improves remote memory function 4 weeks after stroke, compared to vehicle. These results demonstrate that 3K3A-APC might be an effective treatment for white matter stroke.