For the successful treatment of ischemia, it is important to resupply sufficient blood into ischemic regions by inducing angiogenesis. Many stem cell transplantation studies have been reported to enhance angiogenesis, especially those relating to mesenchymal stem cells (MSCs); however cell transplantation has a number of limitations, such as the low rate of cell survival and donor cell shortage. In this study, we developed bioactive peptides by immobilizing substance P into self-assembling peptides, and their MSCs recruiting ability and therapeutic effects were evaluated by using ischemic hind limb models. Limb ischemia was produced in athymic mice, and 1% (wt/vol) peptides were injected into ischemic sites (n = 6 in each group: ischemia, substance P, RADA16-II, RADA16-II + substance P, and RADA16-II + RADA-SP (bioactive peptides)). The tissues were harvested for histological analysis and tissue perfusion measurement at 1, 3, 7, and 28 days after injection. We observed that bioactive peptides assembled themselves (<10 nm nanofibers) and formed 3-dimensional (3D) microenvironments within ischemic regions. In the animal study, it was observed that by applying bioactive peptides, substance P continued to be released at 28 days, and consequently, MSCs were successfully recruited into ischemic regions. Bioactive peptides could prevent fibrosis, promote neovascularization, enhance tissue perfusion, and prevent limb salvages. Our results demonstrated that bioactive peptides are one of the most powerful tools for the treatment of ischemia, through their recruitment of autologous MSCs and promotion of angiogenesis without cells transplantation.