We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-κB (NF-κB) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor κB-α degradation permitting translocation of NF-κB to the nucleus and reporter gene activation. Inhibition of NF-κB activation by overexpression of a nondegradable inhibitor κB-α mutant or treatment with NF-κB inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-κB dependent. These findings support the conclusion that NF-κB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.