In this study, we tissue-engineered heart valves in vivo using autologous bone marrow-derived cells (BMCs). Canine BMCs were differentiated into endothelial cell (EC)-like cells and myofibroblast (MF)-like cells. Decellularized porcine pulmonary valves were seeded with BMCs and implanted to abdominal aorta and pulmonary valve of bone marrow donor dogs. Histological examination of the explants identified the regeneration of valvular structures expressing CD31 and smooth muscle alpha-actin, indicating the presence of EC-like and MF-like cells in the grafts at 3 and 1 week, respectively, after implantation. Fluorescent microscopic examinations identified the presence of fluorescently labeled cells in the explants, indicating that the implanted BMCs survived and participated in the heart valve reconstitution. This study reports, for the first time, on tissue engineering of heart valve in vivo using BMCs.