Andrographolide (Andro), which was isolated from the traditional medicinal herb Andrographis paniculata, has been reported to possess various biological activities. However, its limited potency presents hurdles to its therapeutic development. In this study, the andrographolide derivatives AGP-26a (12β-isomer), AGP-26b (12α-isomer) and AGP-26 (4:1 mixture of AGP-26a and AGP-26b) were synthesized by chemical modification and characterized for their anti-angiogenic properties. We found that AGP-26b could inhibit angiogenesis both in vitro and in vivo with highest efficacy among these three derivatives. Human umbilical vein endothelial cells (HUVECs) and Tg(fli-1a:EGFP)y1 zebrafish model were used to identify the anti-angiogenic activities of AGP-26, AGP-26a, and AGP-26b. The results showed that AGP-26b exhibited the strongest inhibitory activity on VEGF-induced proliferation, migration, invasion and formation of capillary-like structures on HUVECs. In zebrafish model, AGP-26b also showed the strongest suppression of ISV development. Further studies showed that the underlying mechanism of the anti-angiogenic effects of AGP-26b was at least partly through the blockage of VEGF/VEGFR2 signaling pathways. AGP-26b blocked the activation of VEGFR2. Consequently, the phosphorylation of key intracellular proangiogenic kinases such as Src family kinase (Src), focal adhesion kinase (Fak), Mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase 1 and 2 (Erk1/2) and Akt induced by VEGF was all suppressed by the treatment of AGP-26b. Moreover, AGP-26b reduced the protein expression of matrix metalloproteinases (MMP-9 but not MMP-2) in HUVECs. These results provide evidence supporting the notion that AGP-26b may serve as a potential therapeutic v anti-angiogenic agent.