NK cells are innate lymphocytes important for host defense against viral infections and malignancy. However, the molecular programs orchestrating NK cell activation are incompletely understood. MicroRNA-155 (miR-155) is markedly upregulated following cytokine activation of human and mouse NK cells. Surprisingly, mature human and mouse NK cells transduced to overexpress miR-155, NK cells from mice with NK cell-specific miR-155 overexpression, and miR-155(-/-) NK cells all secreted more IFN-γ compared with controls. Investigating further, we found that activated NK cells with miR-155 overexpression had increased per-cell IFN-γ with normal IFN-γ(+) percentages, whereas greater percentages of miR-155(-/-) NK cells were IFN-γ(+). In vivo murine CMV-induced IFN-γ expression by NK cells in these miR-155 models recapitulated the in vitro phenotypes. We performed unbiased RNA-induced silencing complex sequencing on wild-type and miR-155(-/-) NK cells and found that mRNAs targeted by miR-155 were enriched in NK cell activation signaling pathways. Using specific inhibitors, we confirmed these pathways were mechanistically involved in regulating IFN-γ production by miR-155(-/-) NK cells. These data indicate that miR-155 regulation of NK cell activation is complex and that miR-155 functions as a dynamic tuner for NK cell activation via both setting the activation threshold as well as controlling the extent of activation in mature NK cells. In summary, miR-155(-/-) NK cells are more easily activated, through increased expression of proteins in the PI3K, NF-κB, and calcineurin pathways, and miR-155(-/-) and 155-overexpressing NK cells exhibit increased IFN-γ production through distinct cellular mechanisms.