Autologous skin grafts are considered necessary for the treatment of extensive skin defects. However, skin graft by suturing is a time-consuming medical handling and rather stressful event for recipients. To that end, tissue adhesives have been suggested in skin grafts. Chitosan hydrogel is well known as a wound dressing and tissue adhesive material showing biocompatibility, anti-infective activity, and the ability to accelerate wound healing. In this report, we evaluated the application of the chitosan hydrogel as a tissue adhesive in skin grafts. Although chitosan hydrogel shortened the operation time and resulted in a high graft absorption rate in comparison with suturing, wound epithelization was rather retarded. On the other hand, chitosan hydrogel was found more biocompatible than the commonly used tissue adhesive octyl-2-cyanoacrylate. When the chitosan hydrogel was premixed with a serum-free tissue culture medium DMEM/F12, it was found to easily degrade and promote wound epithelization. Histological examination revealed that the medium (DMEM/F12)-containing chitosan hydrogel was associated with the accumulation of polymorphonuclear leukocytes and neovascularization. In addition, immunohistochemical staining showed that the vascular endothelial growth factor (VEGF) was localized in the chitosan hydrogel degraded matrices. And infiltration of leukocytes determined the degradation activity with the D-glucose in the medium (DMEM/F12) suggested to play a central role in chitosan hydrogel degradation. Therefore, the medium (DMEM/F12)-containing chitosan hydrogel may become commonly accepted as a beneficial wound dressing and tissue adhesive in extensive wound management and skin grafts.