Chitosan has a variety of biological functions through conjugating of other compounds to their amino and hydroxyl groups. To further expand applicability of chitosan, we have modified the amino group of chitosan with 2-iminothiolane to bestow thiol groups and obtained about 20% yield, which is equivalent to 913 mu equiv SH/g chitosan or 457 nequiv SH/nmol chitosan. Bovine serum albumin (BSA) was reacted with N-(c-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS), and maleimide-modified BSA (MaINBSA) was obtained. The yield of sulfo-EMCS addition was 12.8-36.8 mol MaIN/mol BSA. When the chitosan-SH was reacted with MalN-BSA via thioether, 97.8% of the maleimide group was reacted, and 37.2% of the SH group was consumed. The remaining SH group was quenched by bromoacetamide. This is the first report of covalent conjugation of a protein to chitosan. Our method should find many applications in developing new chitosan-based biomedical materials containing other components such as growth factors and cell adhesion molecules, known to be crucial to cells. Our thiolated chitosan will facilitate conjugation of such biomedical components to provide new types of materials for tissue engineering.