Integrins are essential protagonists in the complex multistep process of cancer progression and are thus attractive targets for the development of anticancer agents. Cucurbitacin B, a triterpenoid purified from the leaves of Tunisian Ecballium elaterium exhibited an anticancer effect and displayed anti-integrin activity on human glioblastoma U87 cells, without being cytotoxic at concentrations up to 500 nM. Here we show that cucurbitacin B affected the adhesion and migration of U87 cells to fibronectin in a dose-dependent manner with IC50 values of 86.2 nM and 84.6 nM, respectively. Time-lapse videomicroscopy showed that cucurbitacin B significantly reduced U87 cells motility and affected directional persistence. Cucurbitacin B also inhibited proliferation with IC50 value of 70.1 nM using Crystal Violet assay. Moreover, cucurbitacin B efficiently inhibited in vitro human microvascular endothelial cells (HMEC) angiogenesis with concentration up to 10 nM. Interestingly, we demonstrate for the first time that this effect was specifically mediated by α5β1 integrins. These findings reveal a novel mechanism of action for cucurbitacin B, which displays a potential interest as a specific anti-integrin drug.