Abstract Pig-to-human xenotransplantation has the potential to alleviate the shortage of human organs for transplantation. However, rejection remains a hurdle for successful xenograft survival. The first immunological barrier is hyperacute rejection mediated by xenoreactive antibodies and serum complement. An increasing body of evidence indicates that natural killer (NK) cells are involved in the second immunological barrier, the delayed rejection of xenogeneic grafts. NK-mediated cytotoxicity is tightly regulated by signals through activating and inhibitory receptors that recognize HLA class I and unknown ligands on target cells, respectively. In this study, I show that (I) human NK cells mediate xenogeneic cytotoxicity against porcine cells through activating receptors NKG2D and NKp44; (II) pULBP1 serves as the predominant functional ligand for human NKG2D; (III) pULBP1 on porcine endothelial cells (pEC) is tightly regulated by various stimuli; (IV) the level of protection of pEC against human NK cell-mediated cytotoxicity depends both on the expression levels of HLA-E and on the respective NK cell receptor CD94/NKG2A; (V) pEC derived from HLA- E/β2-microglobulin-transgenic pigs are efficiently protected against human NK cell- mediated cytotoxicity. In conclusion, for successful clinical xenotransplantation a crossbreeding of HLA-E/huβ2m-transgenic pigs with other transgenic and knockout pigs (e.g. pULBP1 knockout) may be required to generate pigs resistant to the majority of rejection mechanisms.