Considering the design and development of biomaterials used in tissue engineering, not only is it important that they are biocompatible but also that they induce the desired cellular response for tissue regeneration. Chitosan, a biocompatible and bioresorbable polymer of N-acetylglucosamine and glucosamine is used in our work combined with recombinant human BMP-2 (rhBMP-2), a potentially useful activation factor for bone repair. In this way, we try to combine the biological and filmogenic properties of this biopolymer with the osseoinductive ability of the rhBMP-2. Results showed that the chitosan films employed, without and with rhBMP-2 activation, are able to support cellular growth and proliferation on them and that only the rhBMP-2 activated ones are able to differentiate from a myoblastic mouse cell line (C2C12) toward osteoblastic phenotype. Osseoinduction properties of rhBMP-2 activated films persist for a long storage time. The in vivo experiments performed confirm the expectative created by the in vitro results obtained and are an indication that rhBMP-2 activated chitosan films could be a very attractive biomaterial for the enhancement of osseointegration of surgical prostheses and implants and for the purpose of tissue engineering bone regeneration.