Demineralized bone matrix(DBM) has been reported to have osteoconductive and osteoinductive properties and has been clinically used as a bone graft alternative. In the present study, we generated a vascularized bone flap by subcutaneous implantation of DBM with a vascular loop to provide blood supply in a rat model. Thirty male Sprague-Dawley rats were divided into two groups, according to the presence or absence of blood supply. In the experimental group, the bone flap was created by application of 0.4 ml DBM onto two pieces of gelatin sponge sheets between which a vascular loop was sandwiched. A prefabricated flap without a vascular loop served as the control. The flaps were biopsied at three different time intervals postoperatively (2, 4, and 6 weeks). The results showed that DBM induced subcutaneous bone formation in both of the groups. However, in the non-vascularized group, the amount of bony tissue had decreased at 4 postoperative weeks and continued to do so afterwards. In contrast, bone formation was active at 4 weeks in the vascularized group. Our study indicated that implantation of DBM can prefabricate a bone flap. Blood supply to the flap is considered a key factor to the success of this prefabrication.