The integrity of microvascular endothelium is an important regulator of myocardial contractility. Microvascular barrier integrity could be altered by increased reactive oxygen species (ROS) stress seen within minutes after cardiac arrest resuscitation. Akt and its downstream target NOS3 can protect barrier integrity during ROS stress, but little work has studied these oxidant stress responses in human cardiac microvascular endothelial cells (HCMVEC). We, therefore, studied how ROS affects barrier function and nitric oxide generation via Akt and its downstream target NOS3 in HCMVEC. HCMVEC exposed to H₂O₂ 500 µM had increased Akt phosphorylation within 10 min at both Ser-473 and Thr-308 sites, an effect blocked by the PI3-kinase inhibitor LY294002. H₂O₂ also induced NO generation that was associated with NOS3 Ser-1177 site phosphorylation and Thr-495 dephosphorylation, with Ser-1177 effects attenuated by LY294002 and the Akt inhibitor API-2. H2O2 induced significant barrier disruption in HCMVEC within minutes, but recovery started within 30 min and normalized over hours. The NOS inhibitor L-NAME (200 µM) blocked NO generation but had no effect on H₂O₂-induced barrier permeability or the recovery of barrier integrity. By contrast, the Akt inhibitor API-2 abrogated HCMVEC barrier restoration. These results suggest that oxidant stress in HCMVEC activates NOS3 via Akt. NOS3/NO are not involved in regulation of H₂O₂-affected barrier function in HCMVEC. Independent of NOS3 regulation, Akt proves to be critical for the restoration of barrier integrity in HCMVEC.