Three kinds of cross-linked chitosan films were prepared with hexamethylene diisocyanate (HDI), epichlorohydrin (ECH) and glutaraldehyde (GA) as cross-linking agents, respectively. The physical and mechanical properties, biodegradability and Schwann cell affinity of the cross-linked films were investigated. A significant decrease in the degradation rate in lysozyme solution and a large change in the mechanical properties were observed compared with non-cross-linked chitosan films. The protein adsorption on chitosan films was determined by means of enzyme-linked immunosorbent assay (ELISA). In comparison with the non-cross-linked films, the chitosan films cross-linked with HDI showed a significant increase (up to 40-50%) in both fibronectin and laminin adsorption, while the protein adsorption on the other two kinds of cross-linked films was similar to that on non-crosslinked films. In addition, cell culture revealed that the HDI cross-linked chitosan films enhanced the spread and proliferation of Schwann cells while the other cross-linked films delayed the cell proliferation. These results suggest that HDI cross-linking of chitosan films provides a combination of physical properties, biodegradability and Schwann cell affinity suitable for peripheral nerve regeneration.