The sulfation of a water-insoluble Ganoderma lucidum polysaccharide (GLP) was successfully carried out with chlorosulfonic acid-pyridine in dimethyl formamide to prepare three sulfated GLP derivatives, named sGLP1, sGLP2, and sGLP3. The chemical structure of the sulfated GLP was confirmed by Fourier transform infrared and (13)C NMR analyses. The sGLPs were evaluated for their water solubility, degree of substitution (DS), antioxidant properties, and bile acid-binding capacities. The results showed that sulfation improved the water solubility of GLP and increased its scavenging capacities against hydroxyl and superoxide anion radicals, hydrogen peroxide-scavenging activity, Fe(II) chelating ability, reducing power, and bile acid-binding capacities. It was also observed that the DS may influence the physicochemical and functional properties of sGLPs. For instance, the sulfated GLP with the lowest DS had the greatest bile acid-binding capacity, and the sGLP that had the highest DS showed the lowest bile acid-binding ability under the experimental conditions. The results from this study suggested that sulfation is a possible approach to obtain novel water-soluble derivatives of GLP with improved physicochemical, functional, and biological properties for potential utilization in functional foods or supplemental products.