Thymosin beta4 (Tβ4) has multi-functional roles in angiogenesis and arteriogenesis, but little is known about its mechanism. The Notch signaling pathway is important in regulation of angiogenic behavior of endothelial cells, in addition to vascular endothelial growth factor (VEGF). Whether, Tβ4 regulates angiogenesis through Notch signaling pathway is not clear. In this article, we evaluated the effect of Notch signaling in Tβ4-induced angiogenesis in human umbilical vein endothelial cell (HUVEC). Our results revealed that Tβ4 increased Notch1 and Notch4 expression in a dose and time-dependent manner. The inhibition of Notch1 or Notch4 with siRNA or the Notch receptor inhibitor DAPT significantly prevented Tβ4-induced HUVEC tube formation and lymphocyte transendothelial migration. The inhibition of Notch1 or Notch4 also blocked Tβ4-induced VEGF and HIF-1α expression. VE-cadherin is the major endothelial adhesion molecule in the control of angiogenesis. Tβ4 significantly reduced VE-cadherin expression levels in HUVEC, while the inhibition of Notch signaling prevented Tβ4-induced VE-cadherin down-regulation. The results of this study suggest that Tβ4 induces HUVEC angiogenesis through Notch signaling pathway.