© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Long-term success in pig-to-primate xenotransplantation is currently hampered by acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and injury. Klotho has anti-apoptotic, anti-inflammatory effects on EC and protects EC against reactive oxygen species, rendering klotho a promising molecule to control AVR. In this study, porcine ECs were pre-incubated with klotho and then exposed to xenoreactive antibodies and complement. Real-time PCR revealed that klotho suppressed antibody-induced pro-inflammatory gene expression of VCAM-1 and IL-1α. NF-κB activation, IκBα phosphorylation, was also attenuated by klotho administration. Furthermore, klotho induced in porcine EC resistance against complement-dependent cytotoxicity. Accompanying this change, the binding of IgG and IgM xenoreactive antibodies to porcine EC was decreased and the expression of anti-inflammatory gene HO-1 was upregulated. These findings indicated that klotho protein protected porcine EC from activation and injury caused by binding of xenoreactive antibodies and was a promising candidate molecule in a multitransgenic pig strategy for xenotransplantation.