The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16(+) CD56(-), CD16(-) CD56(+) and CD16(-) CD56(-) cells. NKG2D expression on marmoset CD16(+) CD56(-) and CD16(-) CD56(+) splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16(+) CD56(-) and CD16(-) CD56(+). In addition, CD16(+) CD56(+) cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.