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Timosaponin AⅢ (Timo AⅢ) is a natural steroidal saponin isolated from the traditional Chinese herb Anemarrhena asphodeloides Bge with proved effectiveness in the treatment of numerous cancers.However,whether Timo AⅢ suppresses tumor angiogenesis remains unclear.In the present study,we investigated the antiangiogenesis effects of Timo AⅢ and the underlying mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish embryos in vivo.We showed that treatment with Timo AⅢ (0.5-2 μM) partially disrupted the intersegmental vessels (ISVs) and subintestinal vessels (SIVs) growth in transgenic zebrafish Tg(fli1a:EGFP)y1.Timo AⅢ (0.5-4 μM) dose-dependently inhibited VEGF-induced proliferation,migration,invasion,and tube formation of HUVECs,but these inhibitory effects were not due to its cytotoxicity.We further demonstrated that Timo AⅢ treatment significantly suppressed the expression of VEGF receptor (VEGFR) and the phosphorylation of Akt,MEK1/2,and ERK1/2 in HUVECs.Timo AⅢ treatment also significantly inhibited VEGF-triggered phosphorylation of VEGFR2,Akt,and ERK1/2 in HUVECs.Moreover,we conducted RNA-Seq and analyzed the transcriptome changes in both HUVECs and zebrafish embryos following Timo AⅢ treatment.The coexpression network analysis results showed that various biological processes and signaling pathways were enriched including angiogenesis,cell motility,cell adhesion,protein serine/threonine kinase activity,transmembrane signaling receptor activity,growth factor activity,etc.,which was consistent with the antiangiogenesis effects of Timo AⅢ in HUVECs and zebrafish embryos.We conclude that the antiangiogenesis effect of Timo AⅢ is mediated through VEGF/PI3K/Akt/MAPK signaling cascade;Timo AⅢ potentially exerts antiangiogenesis effect in cancer treatment.