The integrins and PI3K/Akt are important mediators of the signal transduction pathways involved in tumor angiogenesis and cell survival after exposure to ionizing radiation. Selective targeting of either integrins or PI3K/Akt can radiosensitize tumors. In this study, we tested the hypothesis that the combined inhibition of integrin alphanubeta3 by cRGD and PI3K/Akt by LY294002 would significantly enhance radiation-induced inhibition of angiogenesis by vascular endothelial cells. Treatment with cRGD inhibited the adhesion and tube formation of human umbilical vein endothelial cells (HUVECs). The inhibitory effect was further increased when cRGD and LY294002 were applied simultaneously. Both radiation and cRGD induced Akt phosphorylation, up-regulated COX2 expression, and increased PGE2 production in HUVECs. Treatment with LY294002 effectively inhibited radiation- and cRGD-induced Akt phosphorylation and up-regulation of COX2 and increased apoptosis of HUVECs. The combined use of cRGD and LY294002 enhanced radiation-induced cell killing. The clonogenic survival of HUVECs was decreased from 34% with 2 Gy radiation to 4% with these agents combined. These results demonstrate that combined use of ionizing radiation, cRGD and LY294002 inhibited multiple signaling transduction pathways involved in tumor angiogenesis and enhanced radiation-induced effects on vascular endothelial cells.