Reperfusion therapy with recombinant tissue plasminogen activator (rtPA) or mechanical thrombectomy is the most effective treatment for ischemic stroke. However, a large proportion of stroke patients remain severely disabled even after receiving timely reperfusion therapy. It remains unclear how reperfusion therapy results in secondary injury to the brain tissue and whether different reperfusion therapies induce differential effects. Here, we comprehensively determined the spatiotemporal dynamic changes in brain lipids during the acute phase after reperfusion in a mouse model of transient middle cerebral artery occlusion, with or without rtPA administration, using desorption electrospray ionization (DESI)-mass spectrometry imaging (MSI). Several phospholipids, sphingolipids, and neutral lipids were significantly altered both spatially and temporally at multiple timepoints after reperfusion, many of which were closely associated with expansion of the brain infarction territory and neurological function impairment. Furthermore, rtPA treatment significantly increased brain infarction, cerebral edema, and neurological deficits. Consistently, rtPA treatment caused extensive brain lipid alterations by facilitating brain-wide changes in lipid metabolism and inducing ischemic region-specific lipid changes. Overall, these results provide novel insights into how reperfusion therapy affects brain tissue and the outcome of stroke patients, and thus may facilitate the optimization of the treatment of ischemic stroke.