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Marine macroalgae are efficient producers of sulfated polysaccharides. The algal sulfated polysaccharides possess diverse bioactivities and peculiar chemical structures, and represent a great potential source to be explored. In the present study, a heparinoid-active sulfated polysaccharide was isolated from the green alga <i>Cladophora oligoclada.</i> Results of chemical and spectroscopic analyses indicated that the sulfated polysaccharide was composed of →6)-<i>β</i>-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>-(1→, <i>β</i>-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>-(1→, →6)-<i>α</i>-<span style="font-variant: small-caps;">d</span>-Glc<i>p</i>-(1→ and →3)-<i>β</i>-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>-(1→ units with sulfate esters at C-2/C-4 of →6)-<i>β</i>-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>-(1→, C-6 of →3)-<i>β</i>-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>-(1→ and C-3 of →6)-<i>α</i>-<span style="font-variant: small-caps;">d</span>-Glc<i>p</i>-(1→ units. The branches consisting of <i>β</i>-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>-(1→ and →6)-<i>β</i>-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>-(1→ units were located in C-3 of →6)-<i>β</i>-<span style="font-variant: small-caps;">d</span>-Gal<i>p</i>-(1→ units. The sulfated polysaccharide exhibited potent anticoagulant activity in vitro and in vivo as evaluated by activated partial thromboplastin time (APTT), thrombin time, and the fibrinogen level. For the APTT, the signal for clotting time was more than 200 s at 100 μg/mL in vitro and at 15 mg/kg in vivo. The obvious thrombolytic activity of the sulfated polysaccharide in vitro was also found. The mechanism analysis of anticoagulant action demonstrated that the sulfated polysaccharide significantly inhibited the activities of all intrinsic coagulation factors, which were less than 1.0% at 50 μg/mL, but selectively inhibited common coagulation factors. Furthermore, the sulfated polysaccharide strongly stimulated the inhibition of thrombin by potentiating antithrombin-III (AT-III) or heparin cofactor-II, and it also largely promoted the inhibition of factor Xa mediated by AT-III. These results revealed that the sulfated polysaccharide from <i>C. oligoclada</i> had potential to become an anticoagulant agent for prevention and therapy of thrombotic diseases.