The purpose of this study was to evaluate the cell compatibility of collagen-hyaluronan (HA) substrate in vitro by assessing ACL cell cultures. Additionally, the use of collagen-HA substrate as a dressing material for partial ACL defects as well as its effect on collagen synthesis and angiogenesis were evaluated in vivo. The initial attachment and proliferation of dog ACL cells on silk matrix covered with collagen-HA substrate (SMCH) was greater than that observed on silk matrix alone. Silk matrix and SMCHs were implanted as dressing materials into partial ACL defects located at the knees of dogs, and they were harvested six weeks after implantation. A histological evaluation of the collagen-HA substrates revealed the presence of monocytes and the absence of giant cells in all cases. MT staining of the SMCH-grafted group showed a higher level of granulation tissue formation consisting of fibroblasts and collagen fibers compared to the silk matrix-grafted group. In addition, CD31 staining revealed that the SMCH-grafted area showed more blood vessel formation than the silk matrix-grafted area. These results suggest that the collagen-HA substrate was cell-compatible in vitro and enhanced collagen synthesis and new blood vessel formation in vivo.