Traditional Chinese medicine, a rich source of potent cancer chemopreventive agents, is attracting increasing attention worldwide. Recently, the anticancer activity of Trametes robiniophila Μurr. (Huaier) has been widely investigated. However, the mechanisms are not yet fully understood. This study aimed to elucidate the inhibitory effect of Huaier extract on angiogenesis and tumor growth. Incubation with Huaier extract inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) and mouse mammary tumor cells (4T1). In addition, treatment with Huaier extract decreased the motility and tube formation of HUVECs in a dose-dependent manner in vitro. As determined by western blot analysis, Huaier extract dose-dependently decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK), transcription factor p65, c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription 3 (STAT3) and the expression of vascular endothelial growth factor (VEGF). In ex vivo experiments, new vessel growth was suppressed as shown by chick embryo chorioallantoic membrane (CAM) and rat aortic ring assays in the presence of Huaier extract. To further evaluate the inhibitory effect, 4T1 cells were injected subcutaneously into BALB/c mice. The administration of Huaier extract suppressed tumor volume, decreased microvessel density and induced apoptosis. These data suggest that Huaier extract may serve as a potent anti-angiogenic and antitumor agent.