Heparins are widely used in the prevention of primary and/or secondary thrombosis and in patients undergoing cardiopulmonary bypass for heart surgery. The over dosage of unfactionated heparin has to be quickly neutralize by the protamine sulfate (PS). Adverse effects of PS, including allergic reactions, respiratory problems, hypotension, bradycardia and sudden deaths may occur. We synthesized a cationic derivative of dextran modified by the substitution with a glicydyltrimethylamonium chloride (MD). In in vitro studies MD binds to heparin with an efficiency similar to that of PS. The aim of the study was to investigate the neutralization of unfractionated heparin by MD at in vivo conditions. We used the models of FeCl3-induced venous thrombosis (mice) and arterial thrombosis induced by electrical stimulation (rats). The values of thrombus weight and aPTT in the vehicle treated mice were 0.39 ± 0.05 mg and 28.9 ± 1.3 sec., respectively. As expected, administration of heparin resulted in a significant decrease of the thrombus weight (0.01 ± 0.01 mg; p < 0.001) and prolongation of aPTT (300.0 ± 0.0 sec.; p < 0.001). The reversal of heparin action on thrombus weight by MD was observed (0.21 ± 0.03 mg; p < 0.001) even clearer than the effect of PS (0.12 ± 0.01 mg; p < 0.001). MD and PS reversed the prolongation of aPTT (40.7 ± 6.7 sec.; p < 0.01 and 29.4 ± 4.0 sec.; p < 0.01, respectively). The values of thrombus weight, bleeding time and aPTT in the vehicle treated rats were 1.02 ± 0.10 mg, 102.4 ± 2.6 and 20.3 ± 0.9 sec., respectively. Heparin administered in dose of 300 U/kg resulted in a significant decrease of the thrombus weight (0.51 ± 0.06 mg; p < 0.01), prolongation of bleeding time (162.4 ± 9.1 sec.; p < 0.01) (300.0 ± 0.0 sec.; p < 0.001), whereas only bleeding time (144.4 ± 15.4 sec.; p < 0.01) and aPTT (55.5 ± 9.7 sec.; p < 0.001) were significantly changed in rats treated with dose of 150 U/kg. Although MD did not significantly reverse the antithrombotic activity of heparin in rats, it reversed the effect of heparin administered in dose of 300 U/kg on bleeding time (99.6 ± 9.1 sec.; p < 0.01) and aPTT (86.7 ± 17.5 sec.; p < 0.01) and heparin administered in dose of 150 U/kg only on bleeding time (103.5 ± 6.2 sec.; p < 0.05). In contrast to the PS, MD significantly increased red blood cell counts, hemoglobin level and haematocrit value of rats. In conclusion, the MD reduced anticoagulant activity of heparin both under in vitro and in vivo conditions in venous and arterial thrombosis. MD could be a very good substitution of PS in dialyzed patients with the endstage renal disease associated with anemia.