This work is the first to report sulfation of chitosans with different molecular weight (Mw, 51.5-112.4 kDa) via their trimethylsilylated derivatives as the precursors. Different from original chitosan, silylated chitosan was easily sulfated with sulfur trioxide-pyridine complex under homogeneous conditions in DMSO, and highly sulfated products with degree of sulfation (DS) in the range of 1.65-2.46 and Mw in the range of 18.1-54.5 kDa were obtained at 20-40°C. Degradation of chitosan was inevitable in the reaction process. However, the extent of degradation reduced with increasing of degree of trimethylsilylation (DT). The results of UV-vis and fluorescence emission spectra indicated that the chitosan degradation involved elimination reaction. Clotting assays showed that highly sulfated chitosans significantly prolonged activated partial thromboplastin time (APTT) and thrombin time (TT), but not prothrombin time (PT). A high DS (>2.1) value was essential for the anticoagulant activity.