Chitosan is a biodegradable material that has been shown to be biocompatible, and has broad potential for use in tissue engineering. In the present study we generated chitosan derivatives containing anionic groups, and conducted in vitro assays to test their potential as biomaterials for use in tissue engineering. Chitosan derivative containing anionic groups were prepared by reacting the parent compound, chitosan, with propane sultone via a ring-opening reaction. To explore changes in the physicochemical properties of the resulting chitosan derivatives, we analyzed their structures by infrared spectroscopy (IR), proton nuclear magnetic resonance (1H NMR) and wide-angled X-ray diffraction (WAXD). As a result of the anionic sulfonate groups introduced in these molecules, the N-sulfopropylchitosan derivatives showed amphoteric properties. WAXD analysis of the N-sulfopropylchitosan derivatives also showed that their crystallinity decreased with the number of sulfonate moieties introduced on the free amine of chitosan increased. We further examined the biocompatibility of chitosan and the N-sulfopropylchitosan derivatives by investigating their effects on cell growth rate. Our results demonstrate that the cell compatibility of N-sulfoalkylchitosan derivatives with human dermal fibroblasts (HDFs) had dependent on the concentration, and sulfonate moieties. Thus, from the viewpoint of HDF proliferation, there seems to be an optimum moiety of sulfoalkyl groups introduced in chitosan for proliferation of HDFs.