Abstract: In this paper, the anticoagulation approaches of glycosaminoglycan was studied with AT-III-deficient plasma, HC-II-deficient plasma and both factor-deficient plasma in human. The activities of coagulation factor II, V, VII, VIII, IX, X, XI, XII and the fibrinogen content in human plasma were measured using the solidification method. The influence of glycosaminogly could on blood recalcification time in rats was studied by using recalcification experiment. The Ca2 +concentrations in rats and mice serum were measured through methyl thymol blue colorimetric method. The results showed that the antithrombin role of glycosaminoglycan from Urechis unicinctus mainly presented AT- Ⅲ dependency. Glycosaminoglycan could passivate the thrombin inhibitory activity of heparin cofactor II. Glycosaminoglycan significantly reduced the activities of coagulation factor V, VII, X, VIII, IX, XI, XII in the plasma ( p < 0. 05) . Besides, glycosaminoglycan could extend prolong recalcification time in rats by shielding Ca2+in plasma, and significantly reduced Ca2+concentration in rats and mice serum (p<0.01) . Glycosaminoglycan reduced the Ca2+concentration in a more intensive way than that of heparin sodium (p<0.01) .