In rodents, liver natural killer (NK) cells have been shown to mediate higher cytotoxic activity against tumor cells than do peripheral blood (PB) NK cells. However, such differences between liver and PB NK cells have not been extensively investigated in humans. The phenotypical and functional properties of NK cells extracted from liver perfusates at the time of living donor liver transplantation were investigated. The tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL), a critical molecule for NK cell–mediated anti‐tumor cell killing, was not expressed by freshly isolated PB NK cells or by liver NK cells. Stimulation with interleukin (IL)‐2, significantly up‐regulated the expression of TRAIL on liver NK cells, but this effect was barely observed on PB NK cells. Donor liver NK cells showed the most vigorous cytotoxicity against HepG2, a hepatocellular carcinoma (HCC) cell line, after IL‐2 stimulation (90.5% ± 2.2% at E: T = 10:1), compared with donor and recipient PB NK cells and recipient liver NK cells (64.8% ± 8.2%, 56.1% ± 8.9%, and 34.6% ± 7.5%, respectively). IL‐2 stimulation resulted in an increased expression of killing inhibitory receptors on liver NK cells in parallel with TRAIL expression. Consistently, the cytotoxicities of IL‐2–stimulated donor liver NK cells against self and recipient lymphoblasts were negligible. In conclusion, adoptive transfer of IL‐2–stimulated NK cells extracted from donor liver graft perfusate could mount an anti‐tumor response without causing toxicity against 1‐haplotype identical recipient intact tissues. These findings present a concept to prevent recurrence of HCC after liver transplantation. (HEPATOLOGY 2006;43:362–372.)