The role of prostaglandin production in the control of regenerative function of endothelial progenitor cells (EPCs) has not been studied. We hypothesized that activation of cyclooxygenase (COX) enzymatic activity and the subsequent production of prostacyclin (PGI₂) is an important mechanism responsible for the regenerative function of EPCs. In the present study, we detected high levels of COX-1 protein expression and PGI₂ biosynthesis in human EPCs outgrown from blood mononuclear cells. Expression of COX-2 protein was almost undetectable under basal conditions but significantly elevated after treatment with tumor necrosis factor-. Condition medium derived from EPCs hyperpolarized human coronary artery smooth muscle cells, similar to the effect of the PGI₂ analog iloprost. The proliferation and in vitro tube formation by EPCs were inhibited by the COX inhibitor indomethacin or by genetic inactivation of COX-1 or PGI₂ synthase with small interfering (si)RNA. Impaired tube formation and cell proliferation induced by inactivation of COX-1 were rescued by the treatment with iloprost or the selective peroxisome proliferator–activated receptor (PPAR) agonist GW501516 but not by the selective PGI₂ receptor agonist cicaprost. Downregulation of PPAR by siRNA also reduced angiogenic capacity of EPCs. Iloprost failed to reverse PPAR siRNA-induced impairment of angiogenesis. Furthermore, transfection of PGI₂ synthase siRNA, COX-1 siRNA, or PPAR siRNA into EPCs decreased the capillary formation in vivo after transplantation of human EPCs into the nude mice. These results suggest that activation of COX-1/PGI₂/PPAR pathway is an important mechanism underlying proangiogenic function of EPCs.