Black phosphorus (BP), an emerging two-dimensional material with unique optical, thermoelectric, and mechanical properties, has been proposed as bioactive material for tissue engineering. However, its toxic effects on physiological systems remain obscure. The present study investigated the cytotoxicity of BP to vascular endothelial cells. BP nanosheets (BPNSs) with a diameter of 230 nm were fabricated via a classical liquid-phase exfoliation method. Human umbilical vein endothelial cells (HUVECs) were used to determine the cytotoxicity induced by BPNSs (0.31–80 μg/mL). When the concentrations were over 2.5 μg/mL, BPNSs adversely affected the cytoskeleton and cell migration. Furthermore, BPNSs caused mitochondrial dysfunction and generated excessive intercellular reactive oxygen species (ROS) at tested concentrations after 24 h. BPNSs could influence the expression of apoptosis-related genes, including the P53 and BCL-2 family, resulting in the apoptosis of HUVECs. Therefore, the viability and function of HUVECs were adversely influenced by the concentration of BPNSs over 2.5 μg/mL. These findings provide significant information for the potential applications of BP in tissue engineering.