The purpose of this study was to evaluate the effect of collagen-GAG by culturing MSC cell in vitro and to assess ligament regeneration of a collagen-GAG substrate on an ACL reconstruction in an animal model. The silk scaffold was knitted by hand and dry-coated with collagen, while the silk-collagen scaffold was made by covering a silk scaffold with a lyophilized collagen-GAG. The initial attachment and growth of human MSC cells on the silkcollagen scaffold was superior to the attachment and growth observed on the silk scaffold. Silk and silk-collagen scaffolds were implanted in ACLs removed from the knees of rabbits. At 24 weeks after implantation, the silk-collagen scaffold-grafted group showed granulation tissue consisting of fibroblasts and dense collagen fibers. Conversely, no reparative tissues, collagen, or cells were observed in the silk scaffold-grafted group. These results suggest that the lyophilized collagen-GAG substrate increases the adhesion, proliferation, and migration of cells.