The objective of this study was to investigate whether tumor derived fibronectin alternatively spliced EDA domain has a lymphangiogenic potency on human lymphatic endothelial cells (LECs) in tumor generation to facilitate tumor lymphatic metastasis. LECs were cultured in three-dimentional culture system and treated with SW480 supernant which was highly rich in EDA, the result demonstrated that SW480 supernant could facilitate tube-like formations of LECs evidently when compared with controls. Integrinalpha9 was identified by immunofluorescence to be a specific receptor for EDA because we found co-locozation of EDA and integrinalpha9 on LECs as well as significant upregulation of integrinalpha9 in SW480 supernant treated group. Western blot and immunofluorescence revealed that EDA also had important roles accommodating the expressions of some key regulators of lymphangiogenesis such as Prox1 and F-actin so as to facilitate motility and sprouting of LECs. In addition, it had been confirmed that all of these effects could be inhibited markedly by EDA antibody (IST-9). Based on these findings, we assert that EDA derived from tumor cells has an important role in facilitating lymphangiogenesis of malignant tumor. Furthermore, EDA pathway may provide a potent target for tumor lymphatic metastasis therapy.