We examined whether autotransplantation of microvessel fragments (Mvf) and/or myofibroblasts (Mf) into an in vivo skin flap model might improve the survival of the ischemic flap. If so, this could improve blood perfusion, increase blood flow, and improve the survival of the flap. A skin flap was raised on the back of each rat (n = 15 in each group). In the control group, the flap was sutured to the original bed. In the other groups (1) phosphate-buffered saline; (2) autotransplanted Mvf, Mf, or Mvf plus Mf, and (3) a homogenized mixture of Mvf plus Mf was injected into the distal part of the flap. In a further group, Mvf labeled with DiI-acetylated low-density lipoprotein were autotransplanted with Mf into the distal part of the flap, and India ink was perfused through the abdominal aorta 7 days postoperatively. The transplanted Mvf plus Mf group showed better flap survival after 7 days than the other groups (p < 0.02). Labeling with DiI-acetylated low-density lipoprotein showed that transplanted Mvf sent arborizations into the nearby tissue. India ink was found in the lumina within such arborizations. Thus, autotransplanted Mvf may improve the survival of ischemic skin flaps by promoting the early formation of patent connections between Mvf and the host’s microcirculatory system. This apparently requires the presence of Mf.