Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5'-triphosphate RNA (3pRNA) triggers anti-tumor immunity, which is dependent on natural killer (NK) cell activation and cytokine induction. However, to date, RIG-I expression and the functional consequences of RIG-I activation in NK cells have not been examined. Here, we show for the first time the expression of RIG-I in human NK cells and their activation upon RIG-I ligand (3pRNA) transfection. 3pRNA-activated NK cells killed melanoma cells more efficiently than NK cells activated by type I interferon. Stimulation of RIG-I in NK cells specifically increased the surface expression of membrane-bound TNF-related apoptosis-inducing ligand (TRAIL) on NK cells, while activated NK cell receptors were not affected. RIG-I-induced membrane-bound TRAIL initiated death-receptor-pathway-mediated apoptosis not only in allogeneic but also in autologous human leukocyte antigen (HLA) class I-positive and HLA class I-negative melanoma cells. These results identify the direct activation of RIG-I in NK cells as a novel mechanism for how RIG-I can trigger enhanced NK cell killing of tumor cells, underscoring the potential of RIG-I activation for tumor immunotherapy.Keywords: RIG-I; TRAIL; innate immunity; melanoma; natural killer (NK) cells.