We examined the neuroprotective efficacy of docosahexaenoic acid (DHA), an omega-3 essential fatty acid family member, in acute ischemic stroke; studied the therapeutic window; and investigated whether DHA administration after an ischemic stroke is able to salvage the penumbra. In each series described below, SD rats underwent 2 h of middle cerebral artery occlusion (MCAo). In series 1, DHA or saline was administered i.v. at 3, 4, 5, or 6 h after stroke. In series 2, MRI was conducted on days 1, 3 and 7. In series 3, DHA or saline was administered at 3 h, and lipidomic analysis was conducted on day 3. Treatment with DHA significantly improved behavior and reduced total infarct volume by a mean of 40% when administered at 3 h, by 66% at 4 h, and by 59% at 5 h. Total lesion volumes computed from T2-weighted images were reduced in the DHA group at all time points. Lipidomic analysis showed that DHA treatment potentiates neuroprotectin D1 (NPD1) synthesis in the penumbra 3 days after MCAo. DHA administration provides neurobehavioral recovery, reduces brain infarction and edema, and activates NPD1 synthesis in the penumbra when administered up to 5 h after focal cerebral ischemia in rats.