In this study, we evaluated the effect of culture-expanded mesenchymal stem cells (MSCs), derived from amniotic membranes, on neovascularization and blood flow, in an animal model of limb ischemia in immune-deficient mice. MSCs were cultured from human amniotic membranes by collagenase digestion. Human amniotic mesenchymal stem cells (hAMSCs) were administered intramuscularly at three different sites of the ischemic leg whose femoral vessels were ligated. After 4 weeks of culture, a population of homogeneous mesenchymal cells was isolated from the human amniotic membranes after confluence was reached. We performed three different groups of mice model [controls, hAMSCs, conditioned media from the hAMSCs (hAMSCs-CM)]. The blood flow recovery in the hindlimb ischemia model was significantly higher in the hAMSC-transplanted group than in the control group. Moreover, hAMSCs-CM significantly improved the cutaneous blood flow. The histological examination showed that red fluorescence (CM-DiI)-labeled hAMSCs was detected in the interstitial tissues between the muscle fibers 2 weeks after transplantation. The results of this study showed that hAMSCs may be an attractive, alternative source of progenitor or stem cells for basic research as well as clinical applications.