BACKGROUND Autotaxin (ATX-secretory lysophospholipase D) is the primary lysophosphatidic acid (LPA) producing enzyme. LPA promotes endothelial hyper-permeability and microvascular dysfunction following cellular stress. OBJECTIVE We sought to assess whether ATX inhibition would attenuate endothelial monolayer permeability after anoxia-reoxygenation (A-R) in vitro and attenuate the increase in hydraulic permeability observed after ischemia-reperfusion injury (IRI) in vivo. METHODS A permeability assay assessed bovine endothelial monolayer permeability during anoxia-reoxygenation with/without administration of pipedimic acid, a specific inhibitor of ATX, administered either pre-anoxia or post-anoxia. Hydraulic permeability (Lp) of rat mesenteric post-capillary venules was evaluated after IRI, with and without ATX inhibition. Lastly, Lp was evaluated after the administration of ATX alone. RESULTS Anoxia-reoxygenation increased monolayer permeability 4-fold (p < 0.01). Monolayer permeability was reduced to baseline similarly in both the pre-anoxia and post-anoxia ATX inhibition groups (each p < 0.01, respectively). Lp was attenuated by 24% with ATX inhibition (p < 0.01). ATX increased Lp from baseline in a dose dependent manner (p < 0.05). CONCLUSIONS Autotaxin inhibition attenuated increases in endothelial monolayer permeability during A-R in vitro and hydraulic permeability during IRI in vivo. Targeting ATX may be especially beneficial by limiting its downstream mediators that contribute to mechanisms associated with endothelial permeability. ATX inhibitors may therefore have potential for pharmacotherapy during IRI.