Vasculogenic mimicry(VM)is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors.We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2(EphA2)/focal adhesion kinase(FAK)/Paxillin signaling pathways.In this study,we further investigated the anti-VM activity of norcantharidin(NCTD)as a VM inhibitor for gallbladder cancers and the underlying mechanisms.In vivo and in vitro experiments to determine the effects of NCTD on tumor growth,host survival,VM formation of GBC-SD nude mouse xenografts,and vasculogenic-like networks,malignant phenotypes i.e.,proliferation,apoptosis,invasion and migration of GBC-SD cells.Expression of VM signaling-related markers EphA2,FAK and Paxillin in vivo and in vitro were examined by immunofluorescence,western blotting and real-time polymerase chain reaction(RT-PCR),respectively.The results showed that after treatment with NCTD,GBCSD cells were unable to form VM structures when injecting into nude mouse,growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional(3-D)matrix,proliferation,apoptosis,invasion,migration of GBC-SD cells were affected;and survival time of the xenograft mice was prolonged.Furthermore,expression of EphA2,FAK and Paxillin proteins/mRNAs of the xenografts was downregulated.Thus,we concluded that NCTD has potential antiVM activity against human gallbladder cancers;one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway. Vasculogenic mimicry (VM) is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2) / focal adhesion kinase (FAK) / Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD) as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, Host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes ie, proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed that after treatment with NCTD, GBCSD cells were unable to fo rm VM structures when injected into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D) matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected ; and survival time of the xenograft mice was prolonged. Stillrther, expression of EphA2, FAK and Paxillin proteins / mRNAs of the xenografts was downregulated.Thus, we concluded that NCTD has potential antiVM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2 / FAK / Paxillin signaling pathway.