Lineage differentiation and the formation of heterogeneous mature subsets are crucial for immune cells to maintain a breadth of responsiveness to pathogens while controlling reactivity to self. In this study, we report that CD27 is a key marker of the NK cell lineage, dissecting the mature Mac-1high NK cell pool into two functionally distinct subsets. The CD27low NK cell subset possesses a higher threshold to stimulation and appears to be tightly regulated by the expression of NK cell inhibitory receptors. Comparatively, the CD27high NK cell subset displays a greater effector function, exhibits a distinct tissue distribution and responsiveness to chemokines, and interacts productively with dendritic cells. Importantly, we have verified that CD27high and CD27low subsets with distinct cell surface phenotypes also exist in human peripheral blood. These findings clearly reclassify mature NK cells into two distinct subsets and begin to discern their specific role in immune responses.