Umbilical cord blood (CB) represents a main source of circulating endothelial progenitor cells (cEPCs). In view of their clinical use, in either the autologous or allogeneic setting, cEPCs should likely be expanded from CB kept frozen in CB banks. In this study, we compared the expansion, functional features, senescence pattern over culture, and in vivo angiogenic potential of cEPCs isolated from fresh or cryopreserved CB (cryoCB). cEPCs could be isolated in only 59% of cryoCB compared to 94% for fresh CB, while CB units were matched in terms of initial volume, nucleated and CD34(+) cell number. Moreover, the number of endothelial colony-forming cells was significantly decreased when using cryoCB. Once cEPCs culture was established, the proliferation, migration, tube formation, and acetylated-LDL uptake potentials were similar in both groups. In addition, cEPCs derived from cryoCB displayed the same senescence status and telomeres length as that of cEPCs derived from fresh CB. Karyotypic aberrations were found in cells obtained from both fresh and cryoCB. In vivo, in a hind limb ischemia murine model, cEPCs from fresh and cryoCB were equally efficient to induce neovascularization. Thus, cEPCs isolated from cryoCB exhibited similar properties to those of fresh CB in vitro and in vivo. However, the low frequency of cEPCs colony formation after cryopreservation shed light on the need for specific freezing conditions adapted to cEPCs in view of their future clinical use.