Natural killer (NK) cells play key roles in immune surveillance against tumors and viral infection. NK cells distinguish abnormal cells from healthy cells by cell-cell interaction with cell surface proteins and then attack target cells via multiple mechanisms. In addition, extracellular vesicles (EVs) derived from NK cells (NK-EVs), including exosomes, possess cytotoxic capacity against tumor cells, but their characteristics and regulation by cytokines remain unknown. Here, we report that EVs derived from human NK-92 cells stimulated with IL-15 + IL-21 show enhanced cytotoxic capacity against tumor cells. Major cytolytic granules, granzyme B and granzyme H, are enriched by IL-15 + IL-21 stimulation in NK-EVs; however, knockout experiments reveal those cytolytic granules are independent of enhanced cytotoxic capacity. To find out the key molecules, mass spectrometry analyses were performed with different cytokine conditions, no cytokine, IL-15, IL-21, or IL-15 + IL-21. We then found that CD226 (DNAM-1) on NK-EVs is enriched by IL-15 + IL-21 stimulation and that blocking antibodies against CD226 reduced the cytolytic activity of NK-EVs. We also show NK-EVs are taken up by target cells via macropinocytosis. Collectively, our findings elucidate the novel properties of NK-EVs and the mechanism of their incorporation into target cells.