In a search for the wound healing accelerators, we found that tetraacetyl-phytosphingosine (TAPS), a sphingolipid metabolite produced by phytosphingosine acetylation, has significant inhibitory potential on healing of rabbit ear wound. As angiogenesis is fundamental to proper wound healing, we examined the effect of TAPS on angiogenesis using human umbilical vein endothelial cells cultured in vitro. TAPS markedly decreased vascular endothelial growth factor (VEGF)-induced chemotactic migration and capillary-like tube formation. Recognizing its inhibitory potential on angiogenesis, we further investigated the action mechanism of TAPS. TAPS significantly inhibited VEGF-induced proteolytic enzyme production, including matrix metalloproteinase-2, urokinase-type plasminogen activator and plasminogen activator inhibitor-1. TAPS also suppressed VEGF-induced phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In addition, TAPS abolished VEGF-induced intracellular calcium increase, measured using laser scanning confocal microscopy. Together, these results suggest that TAPS exerts its inhibitory action on angiogenesis through the inhibition of mitogen-activated protein kinase activation and intracellular calcium increase, thereby affecting the process of wound healing negatively.