Vascular endothelial growth factor (VEGF) is a pleiotropic factor that regulates embryonal vasculogenesis, tumor angiogenesis and vascular permeability. Among eight differential isoforms, VEGF 121 mainly regulates vascular permeability, while VEGF 165 induces angiogenesis. Our previous studies have suggested that VEGF 121 and VEGF 165 are mainly detected in the lesions of psoriasis and atopic dermatitis, especially VEGF 121. VEGF 121 is the most predominant isoform, which plays a major role in the increased vascular permeability in the aforementioned skin lesions. Thus, the differential expression of VEGF isoforms may be critical in determining either an angiogenic or a hyper-permeable state. However, the distinct VEGF signaling pathways that induce angiogenesis and vascular hyper-permeability in endothelial cells have never been demonstrated. To clarify the differential effects elicited by VEGF 121 and VEGF 165, we compared the biological responses and the signaling pathways activated by VEGF 121 and VEGF 165 in human umbilical vein endothelial cells (HUVEC). VEGF 165 significantly increased the level of phosphorylation in the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, whereas VEGF 121 had little to no effect. In contrast, VEGF 121 induced rapid activation of the Src pathway, while VEGF 165 showed a less pronounced and delayed activation of the Src pathway. Furthermore, the VEGF-induced hyper-permeability and cell proliferation of HUVEC were inhibited by a Src inhibitor (PP2) and a MEK inhibitor (PD98059), respectively. These results indicate that distinct signaling pathways confer different vascular responses to VEGF 121 and VEGF 165.