In order to develop new anticoagulants as the potential alternatives of heparin, microcrystalline cellulose (MCC) was used as a starting polymer and chlorosulfonic acid in N,N-dimethylformamide (ClSO3H-DMF) was used as a sulfation agent for the semi-synthesis of cellulose sulfate sodium, coded as Na-MCS. IR spectra showed that the sulfate group was introduced by the reaction. The sulfation degree (DS) was estimated by means of elemental analysis. 13CNMR indicated that sulfation occurred mainly at C6, partially at C2 and no substitution at C3. Na-MCS with DS of 1.70 was selected for anticoagulant activity investigation. The anticoagulant efficacy was evaluated by coagulation assays and the anticoagulant mechanism was further disclosed by inhibitory analysis of the coagulation factors using chromogenic substrates. Results showed that 1mmol/L Na-MCS could significantly prolongAPTT and TT, but less effect on PT. It represented potent anticoagulant in vitro reaching the efficacy of heparin (150IU/mg) in a certain range of concentration. Further investigation indicated that Na-MCS had a potent anticoagulant activity due to the inhibition of IIa & Xa activities by antithrombinAT III.