A key element for the physiological restriction of blood coagulation at the endothelial cell surface is its non-thrombogenic property, mainly attributed to cell surface heparan sulfate proteoglycans. Heparanase is an endo-beta-D-glucuronidase with specific heparan sulfate degrading activity, which is produced and stored in platelets, and is released upon their activation. We examined the effects of heparanase pro-enzyme on coagulation functions, predominantly under physiological conditions. While heparanase pro-enzyme does not directly affect coagulation protein activities, it has profound effects on heparinoid-mediated regulation of coagulation responses, apparently via mechanisms that do not involve its enzymatic activity. Heparanase pro-enzyme reverses the anti-coagulant activity of unfractionated heparin on the coagulation pathway as well as on thrombin activity. In addition, heparanase pro-enzyme abrogated the factor X inhibitory activity of low-molecular-weight heparin (LMWH). The pro-coagulant effects of the non-active heparanase were also exerted by its major functional heparin-binding peptide. Finally, the effects of heparanase on the activity of factor VII activating protease that is auto-activated by heparinoids indicated a complete antagonistic action of heparanase in this system. Altogether, heparanase pro-coagulant activities that were also demonstrated in plasma samples from patients under LMWH treatment, point to a possible use of this molecule as antagonist for heparinoid treatment.