Abstract: A new corncob xylan sulfate(XS) was synthesized by sulfur trioxide-pyridine(SO₃·Py) method with corncob xylan extracted by alkali method as raw material and LiCl as catalyst in aprotic solvent. The influences of solvent, temperature, reaction time and material ratio on the degree of substitution(D_S) of XS were investigated and the optimal conditions were determined by orthogonal test. The structures and properties of sulfated polysaccharides were investigated by GPC, FT-IR, elemental analysis and ¹³C NMR spectroscopy. And the in vitro anticoagulant activity of XS was studied.The results indicated that the optimal conditions for sulfation were DMF as solvent, molar ratio of SO₃·Py complex and hydroxyl unit of xylose 1.5:1, 55℃ and 3 h, and the degree of substitution of XS was 1.53 and the yield of XS was 78.2%. And the weight average molecular mass of the xylan sulfate product was 36 754 and the dispersion coefficient was 1.191. Structural characterization revealed that sulfate groups were successfully introduced into xylan. A preliminary study of vitro anticoagulant activity was conducted and the results showed that the xylan sulfate had anticoagulant activity, which could significantly prolong the APTT and TT. When the degree of substitution was 1.53 and the mass concentration was 20 mg/L, the APTT, PT and TT of XS were 36.37, 14.22 and 14.70 s, respectively, which were basically equivalent to the positive control heparin sodium.