Mesenchymal stromal/stem cells derived from human Wharton's jelly (WJ-MSC) have emerged as a favorable source for autologous and allogenic cell therapy. Here, we characterized the proangiogenic features of WJ-MSCs and examined their ability to form functional vessels in in vivo models. First, we examined whether WJ-MSCs express endothelial and smooth muscle cell specific markers after culture in endothelial growth media. WJ-MSCs expressed an endothelial specific marker, VEGFR1, at mRNA and protein levels, but did not express other specific markers (VEGFR2, Tie2, vWF, CD31, and VE-cadherin). Rather, WJ-MSCs expressed smooth muscle cell specific markers, α-SMA, PDGFR-β and calponin, and were unable to form tube-like structures with lumen on Matrigel. WJ-MSCs secreted growth factors including angiogenin, IGFBP-3, MCP-1, and IL-8, which stimulated endothelial proliferation, migration, and tube formation. When WJ-MSCs suspended in Matrigel were implanted into nude mice, it led to formation of functional vessels containing erythrocytes after 7 days. However, implantation of endothelial cell-suspended Matrigel resulted in no perfused vessels. The implanted WJ-MSCs were stained positively for calponin or PDGFR-β and were located adjacent to the lining of mouse endothelial cells that were stained with labeled BS-lectin B4. In a murine hindlimb ischemia model, the transplantation of MSCs (5×10(5)cells) into the ischemic limbs improved perfusion recovery and neovascularization of the limbs compared to control group. Therefore, the results suggest that WJ-MSCs promote neovascularization and perfusion by secreting paracrine factors and by functioning as perivascular precursor cells, and that WJ-MSCs can be used efficiently for cell therapy of ischemic disease.