Considering NK cells are an important innate lymphocyte in host protection with their cytotoxic effector functions, it is important to understand how NK cell homeostasis and effector functions can be maintained in peripheral tissues. In this study, the importance of protein phosphatase 2A (PP2A) as a key negative regulator in maintaining NK cell homeostasis and effector functions will be discussed. In vivo treatment of PP2A-specific inhibitors (PP2Ai) differentially modified NK cells in the lymphoid tissues and peripheral tissues. In the peripheral tissues, PP2Ai treatment initially induced the premature CD11b^hi CD27^hi NK cell proliferation, followed by an increase in the terminally differentiated mature CD11b^hi CD27^lo NK subset. Accordingly, the Ki-67⁺ bone marrow NK cells were mobilized to the peripheral tissues upon PP2Ai treatment. Mechanistically, the upregulation of the c-Myc pathway and its phosphorylation, along with its downstream cyclin E expression and G1/S cell cycle transition, was observed in the NK cells of PP2Ai-treated mice. In PP2Ai-treated mice, the expressions of T-bet and its downstream effector molecules, granzyme B and IFN-γ, were also upregulated in NK cells. PP2Ai treatment increased the phosphorylation of mTOR and S6 in NK cells, and an mTOR inhibitor abrogated the effects of PP2Ai in NK cells. Importantly, NK cells isolated from PP2Ai-treated mice showed higher cytotoxicity and IFN-γ production; therefore, they increased the anti-tumor effector function of NK cells. Collectively, these results suggest that PP2A acts as a key negative regulator of NK cell homeostasis and effector functions by modulating both c-Myc- and mTOR-dependent regulatory mechanisms.