We have investigated the effects of bufalin, one of the prominent components in Chinese toad venom, on proliferation of bovine aortic endothelial (BAE) cells and tube formation in three-dimensional type I collagen matrix. In the present study, bufalin potently inhibited the formation of capillary-like tubular networks in a dose-dependent manner. Bufalin also inhibited the proliferation of BAE cells at the same concentration (5 nM) that the tube formation was inhibited. As a potent inhibitor of endothelial cell proliferation, bufalin specifically prevented the entry of BAE cells into the G0/G1 phase of a cell cycle. These findings suggest that in vitro angioinhibitory action of bufalin may be induced by the proliferation inhibition of endothelial cells through the arrest at the G2/M phase of a cell cycle.