Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalisation has been implicated in VEGFR2 signalling, but its role in the activation of ERK1/2 is unclear. In order to explore this question we utilised pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalisation of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signalling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalisation is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalisation is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalisation also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalisation may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signalling in human ECs and targeting receptor trafficking may present a therapeutic opportunity to block tumour angiogenesis.