Xenotransplantation is a promising alternative to overcome donor shortage in transplantation. CD40 molecule plays an important role in the interaction of T cells with antigen-presenting cells and in the activation of vascular endothelial cells. We investigated whether the xenogeneic interaction between human CD40L (hCD40L) on T cells and porcine endothelial CD40 (pCD40) can activate porcine endothelial cells (PECs). The interaction between hCD40L and pCD40 induced the expression of chemokines on PECs as well as MHC and adhesion molecules. Furthermore, NF-kappaB signaling was activated in HEK 293 cells expressing pCD40 and PECs by stimulation with hCD40L+ Jurkat T clones. Both anti-CD40L neutralizing antibodies and NF-kappaB signal inhibitors interfered with immune activation of PECs. Overall, this study shows that xenogeneic interaction between hCD40L and pCD40 can activate PECs through NF-kappaB signaling, and therefore may contribute to acute vascular rejection in xenotransplantation.