When encountering target cells, NK (natural killer) cells exocytose Pfn (perforin) and granzyme B to kill challengers. We previously reported that granzyme B is recycled and reused by NK cells via clathrin-dependent endocytosis. However, whether Pfn, a main secretory vesicle content, indispensible to granzyme B killing, undergoes endocytosis remains unknown. We demonstrate that Pfn is recaptured by early endosomes of NK cells via a clathrin-dependent endocytosis after target cell stimulation. Inhibition of clathrin-dependent endocytosis significantly attenuated the cytotoxicity of NK cells. The data suggest that the recovery of Pfn contributes to the cytotoxicity of NK cells. The assay of endocytosis of lytic molecule presents a particular focus for exploring the mechanism of abnormal cytotoxicity of NK cells.