Fascaplysin is a natural product isolated from marine sponges that exhibits broad anticancer activity. Previous studies revealed that fascaplysin-induced apoptosis and angiogenesis inhibition in vascular endothelial cells contributed to its anticancer activity. Accumulating evidence indicates that autophagy plays a significant role in mediating the function of vascular endothelial cells (VECs) and the response to cancer therapy. However, the effect of fascaplysin on VEC autophagy and the role of autophagy in fascaplysin-induced vascular endothelial cell apoptosis and angiogenesis inhibition are not clear. The present study found that fascaplysin induced autophagy in vascular endothelial cells. Suppression of autophagy using a pharmacological inhibitor (3-methyladenine) or RNA interference of an essential autophagy gene (ATG5) enhanced the cell death and anti-angiogenesis activity of fascaplysin. We further found that fascaplysin significantly increased p8 protein and reactive oxygen species (ROS) levels and decreased mitochondrial membrane potential but had no effect on the mTOR pathway in VECs. Notably, the ROS scavenger N-acetylcysteine inhibited fascaplysin-induced autophagy and increased p8 protein level. Knockdown of p8 by using RNA interference inhibited the autophagy but increased the level of ROS in VECs. Taken together, these data indicated that fascaplysin activated autophagy as a cytoprotective response via ROS and p8 in VECs. Our findings provided important insight into the response of VECs to fascaplysin and may be useful for improving the anticancer efficacy of fascaplysin.