Ischemic stroke causes brain endothelial cell death and damages tight junction integrity of the blood-brain barrier (BBB). We engineered endothelial cell-derived extracellular vesicles (EVs) for the delivery of exogenous heat shock protein 27 (HSP27) and harnessed the innate EV mitochondrial load as a one, two-punch strategy to increase brain endothelial cell survival (via mitochondrial delivery) and preserve their tight junction integrity (via HSP27 delivery). We demonstrated that endothelial microvesicles but not exosomes transferred their mitochondrial load that subsequently underwent fusion with the mitochondrial network of the recipient primary human brain endothelial cells. This mitochondrial transfer increased the relative ATP levels and mitochondrial function in the recipient endothelial cells. EV-mediated HSP27 delivery to primary human brain endothelial cells decreased the paracellular permeability of small and large molecular mass fluorescent tracers in an in vitro model of ischemia/reperfusion injury. This one, two-punch approach to increase the metabolic function and structural integrity of brain endothelial cells is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.Highlights➢Exosomes and microvesicles (EVs) can be engineered for co-delivery of bio-actives➢Microvesicles (MV) but not exosomes contain functional mitochondria➢MV mitochondria fused with the mitochondria in recipient brain endothelial cells➢MVs increase mitochondrial function while EVs increase cellular ATP levels➢EV-mediated HSP27 delivery decreased dextran permeability in brain endothelial cellsGraphical Abstract