Here we report on the existence and functionality of the immune checkpoint signal regulatory protein α (SIRPα) in NK cells and describe how it can be modulated for cell therapy. NK cell SIRPα is up-regulated upon IL-2 stimulation, interacts with target cell CD47 in a threshold-dependent manner, and counters other stimulatory signals, including IL-2, CD16, or NKG2D. Elevated expression of CD47 protected K562 tumor cells and mouse and human MHC class I-deficient target cells against SIRPα+ primary NK cells, but not against SIRPα- NKL or NK92 cells. SIRPα deficiency or antibody blockade increased the killing capacity of NK cells. Overexpression of rhesus monkey CD47 in human MHC-deficient cells prevented cytotoxicity by rhesus NK cells in a xenogeneic setting. The SIRPα-CD47 axis was found to be highly species specific. Together, the results demonstrate that disruption of the SIRPα-CD47 immune checkpoint may augment NK cell antitumor responses and that elevated expression of CD47 may prevent NK cell-mediated killing of allogeneic and xenogeneic tissues.