Aim Increased thromboxane A2 and peroxynitrite are hallmarks of cerebral ischaemia/reperfusion (I/R). Stimulation of thromboxane/prostaglandin receptors (TP) attenuates endothelium‐derived hyperpolarization (EDH). We investigated whether EDH‐type middle cerebral artery (MCA) relaxations following TP stimulation are altered after I/R and the influence of peroxynitrite. Methods Vascular function was determined by wire myography after TP stimulation with the thromboxane A2 mimetic 9,11‐dideoxy‐9α, 11α ‐methano‐epoxy prostaglandin F2α (U46619) in MCA of Sprague Dawley rats subjected to MCA occlusion (90 min)/reperfusion (24 h) or sham operation, and in non‐operated (control) rats. Some rats were treated with saline or the peroxynitrite decomposition catalyst 5,10,15,20‐tetrakis(4‐sulfonatophenyl)porphyrinato iron (III) (20 mg kg−1). Protein expression was evaluated in MCA and in human microvascular endothelial cells submitted to hypoxia (overnight)/reoxygenation (24 h) (H/R) using immunofluorescence and immunoblotting. Results In U46619‐pre‐constricted MCA, EDH‐type relaxation by the proteinase‐activated receptor 2 agonist serine–leucine–isoleucine–glycine–arginine–leucine–NH2 (SLIGRL) was greater in I/R than sham rats due to an increased contribution of small‐conductance calcium‐activated potassium channels (SKCa), which was confirmed by the enlarged relaxation to the SKCa activator N‐cyclohexyl‐N‐2‐(3,5‐dimethyl‐pyrazol‐1‐yl)‐6‐methyl‐4‐pyrimidinamine. I/R and H/R induced endothelial protein tyrosine nitration and filamentous‐actin disruption. In control MCA, either cytochalasin D or peroxynitrite disrupted endothelial filamentous‐actin and augmented EDH‐type relaxation. Furthermore, peroxynitrite decomposition during I/R prevented the increase in EDH‐type responses. Conclusion Following TP stimulation in MCA, EDH‐type relaxation to SLIGRL is greater after I/R due to endothelial filamentous‐actin disruption by peroxynitrite, which prevents TP‐induced block of SKCa input to EDH. These results reveal a novel mechanism whereby peroxynitrite could promote post‐ischaemic brain injury.