Our study investigates the features and anti-tumor function of extracellular vesicles (EVs) isolated from our well described NK cell line - NK3.3 - the only published clonal, normal human cell line to date. Natural killer (NK) cells, critical for proper immune function, destroy tumor cells primarily through the release of granules containing potent cytolytic molecules. NK cells also release these molecules within membrane-bound exosomes and microvesicles collectively known as EVs. We show that NK3.3 EVs contain the cytolytic molecules perforin, granzymes A and B, and granulysin, and an array of common EV proteins. We previously showed that the E3 ubiquitin ligase identified by our laboratory, natural killer lytic-associated molecule (NKLAM/RNF19b), is localized to NK granules and is essential for maximal NK killing; our current study shows NKLAM situated within the NK3.3 EV membrane. We demonstrate that treatment of an array of hematopoietic and non-hematopoietic tumor cell lines with NK3.3 EVs inhibits proliferation and induces apoptosis and cell death while leaving normal cells unaffected. The anti-tumor effects of NK3.3EVs were evaluated via proteomic analysis of K562 leukemia cells treated with NK3.3-derived EVs compared to treatment with non-cytotoxic HEK293 cell-derived EVs or PBS. The results of this analysis suggest strong inhibitory regulation of cell cycle- and cell survival-associated proteins. Taken together, our study suggests that NK3.3 EVs have the potential to be effective immunotherapeutic agents.