To characterize the protein expression profiles and identify the molecules associated with tumor angiogenesis, the cellular proteins of human umbilical vein endothelial cells (HUVECs) in response to cancer cell-conditioned medium (CM) prepared from HT1080 human fibrosarcoma cells were analyzed using fluorescence-labeled 2D gel-based proteomics. Most differentially expressed proteins in HT1080-CM-stimulated cells were found to be downregulated (88%) rather than upregulated (12%) based on statistical analysis of protein spot signals. Additionally, we examined the effects of vascular endothelial cell growth factor (VEGF), a proangiogenic factor, on cellular protein expression. In contrast, most differentially expressed proteins were found to be upregulated (59%) rather than downregulated (41%) in VEGF-stimulated HUVECs. Comparative analyses of 29 and 35 protein species identified in CM-stimulated and VEGF-stimulated HUVECs, respectively, revealed the remarkable differences between these two stimulations. Only four proteins were differentially expressed by both treatments: annexin A2, enolase 1, and T-plastin (downregulated by CM but upregulated by VEGF), and RAN (downregulated by both CM and VEGF). These findings provide new information regarding the regulation of protein expression associated with tumor angiogenesis.