NK cells have been increasingly reported to be an important effector in autoimmune diseases. However, nothing is known in this regard in DED, the most common eye pathology, which is characterized by sustained inflammation on the ocular surface. In the present study, we have examined the profile of NK cells on the ocular surface as well as in the draining lymphoid tissues during the development of this disease. Our data demonstrate activated NK cells during the disease-induction phase. Moreover, in vivo depletion of NK cells in mice results in reduced disease severity and diminished proinflammatory cytokines. Furthermore, we show that NK cells are also able to modulate the maturation of APCs, which is correlated with IFN-γ from NK cells. Together, our findings provide new in vivo evidence that IFN-γ-secreting NK cells can promote induction of DED via direct target tissue damage and indirect influence on the priming phase of an adaptive immune response in secondary lymphoid tissue.