This study investigated the effects of 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid (IOX3), a selective small molecule inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylases, on mouse brains subject to transient focal cerebral ischaemia. Male, 8-to 12-week-old C57/B6 mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) either immediately or 24 h after receiving IOX3. Mice receiving IOX3 at 20 mg/kg 24 h prior to the MCAO had better neuroscores and smaller blood-brain barrier (BBB) disruption and infarct volumes than mice receiving the vehicle, whereas those having IOX3 at 60 mg/kg showed no significant changes. IOX3 treatment immediately before MCAO was not neuroprotective. IOX3 up-regulated HIF-1a, and increased EPO expression in mouse brains. In an in vitro BBB model (RBE4 cell line), IOX3 up-regulated HIF-1a and delocalized ZO-1. Pre-treating IOX3 on RBE4 cells 24 h before oxygenglucose deprivation had a protective effect on endothelial barrier preservation with ZO-1 being better localized, while immediate IOX3 treatment did not. Our study suggests that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of BBB protection, while immediate application could be detrimental. These results provide information for studies aimed at the therapeutic activation of HIF pathway for neurovascular protection from cerebral ischaemia. Keywords: BBB, EPO, HIF, IOX3, preconditioning, stroke. J. Neurochem. (2014) 131, 177-189.The hypoxia-inducible factors (HIFs) are transcription factors that regulate gene expression in response to cellular hypoxia (Semenza 1999;Pugh and Ratcliffe 2003;Kaelin and Ratcliffe 2008;Greer et al. 2012). A mechanism by which the human HIFs 'sense' oxygen levels is enabled by four oxygen-sensitive hydroxylases: three prolyl hydroxylases (PHDs) and one asparaginyl hydroxylase, factor inhibiting HIF (FIH) (Schofield and Ratcliffe 2004). In normoxia, hydroxylation of either of two prolyl residues in the oxygen-dependent degradation domains of HIF-1a promotes its interaction with the von Hippel-Lindau ubiquitin E3 ligase complex. Thereafter, HIF-1a is targeted