It has been studied that mesenchymal stem cells (MSCs) have the capability to promote angiogenesis. Furthermore, there is strong evidence that hypoxic conditions can enhance angiogenesis and immune modulation mediated by MSCs, a notion that has been applied in many fields of clinical application. In the present study, we compared the efficacy of hypoxia preconditioned human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and normoxia conditioned hUC-MSCs for the treatment of ischemic injury in hindlimbs of an immunodeficient mouse model. Expression of negative markers for MSC such as CD31, CD34, and CD45 or positive markers such as CD44, CD73, CD90, and CD105 was not significantly changed in hypoxia preconditioned hUC-MSCs compared with hUC-MSCs cultured in normoxic condition. Expression of angiogenesis-related genes such as COX-2, VEGF, Tie-2, and TGF-β1 was increased compared with hUC-MSCs cultured in normoxic conditions. In the in vivo model, CD31 expression as a marker of angiogenesis was significantly increased in the ischemic limbs at 1 month after injection with hypoxic hUC-MSCs. Angiogenesis-related genes such as Ang-1, COX-1, PIGF, and MCP-1 were significantly upregulated in the muscle of ischemic hindlimbs treated with hypoxic hUC-MSCs than normoxic hUC-MSCs. Expression of proinflammatory genes such as IL-1, and IL-20 was reduced, whereas TGF-β1, which has an anti-inflammatory effect, was strongly increased. In conclusion, hypoxic culture conditions could induce expression of angiogenesis related genes in hUC-MSCs, and hypoxia preconditioned hUC-MSCs showed enhancing effects by inducing angiogenesis and low inflammatory immune response compared with normoxic hUC-MSCs in the ischemia injured hindlimb of immunodeficient mice.