It has been reported that histidine-rich protein II (HRPII), secreted by the malaria parasite, Plasmodium falciparum (Pf), inhibits the heparin-dependent anticoagulant activity of antithrombin (AT) in vitro and in plasma-based assay systems.,The objective of this study was to test the hypothesis that HRPII may also interact with the AT-binding vascular glycosaminoglycans (GAGs), thereby inhibiting the anti-inflammatory signaling function of the serpin.,We expressed HRPII in bacteria, purified it to homogeneity and studied its effect on endothelial cell signaling in the absence and presence of AT employing established signaling assays.,We demonstrate that a low concentration of HRPII potently disrupts the barrier permeability function of endothelial cells. Moreover, HRPII competitively inhibits the protective effect of AT by a concentration-dependent manner. Similarly, AT inhibits the pro-inflammatory activity of HRPII by a concentration-dependent manner. The siRNA knockdown of 3-O-sulfotransferase 1 (3-OST-1), the enzyme responsible for the essential 3-O-sulfation of the AT-binding GAGs, downregulates the pro-inflammatory function of HRPII in endothelial cells, supporting the hypothesis that HRPII competitively inhibits the interaction of AT with 3-OS containing vascular GAGs. Histidine-rich protein II elicits its barrier-disruptive effect by the Src-dependent phosphorylation of vascular endothelial (VE)-cadherin and AT counteracts this effect. We further demonstrate that inorganic polyphosphates bind HRPII with a high affinity to amplify the pro-inflammatory signaling function of HRPII in both cellular and in vivo permeability models.,We postulate that Pf-derived HRPII and polyphosphate can contribute to the pathogenesis of malaria infection by downregulating the AT-dependent anti-inflammatory and anticoagulant pathways.,© 2019 International Society on Thrombosis and Haemostasis.