NKR-P1B is a homodimeric type-II transmembrane C-type lectin-like receptor that inhibits natural killer (NK) cell function upon interaction with its cognate C-type lectin-related ligand, Clr-b. The NKR-P1B:Clr-b interaction represents an MHC-I-independent missing-self recognition system that monitors cellular Clr-b levels. We have generated NKR-P1BB6-deficient (Nkrp1b-/-) mice to study the role of NKR-P1B in NK cell development and function in vivo. NK cell inhibition by Clr-b is abolished in Nkrp1b-/- mice, confirming the inhibitory nature of NKR-P1BB6. Inhibitory receptors also promote NK cell tolerance and responsiveness to stimulation; hence, NK cells expressing NKR-P1BB6 and Ly49C/I display augmented responsiveness to activating signals versus NK cells expressing either or none of the receptors. In addition, Nkrp1b-/- mice are defective in rejecting cells lacking Clr-b, supporting a role for NKR-P1BB6 in MHC-independent missing-self recognition of Clr-b in vivo. In contrast, MHC-I-dependent missing-self recognition is preserved in Nkrp1b-/- mice. Interestingly, spontaneous myc-induced B lymphoma cells may selectively utilize NKR-P1B:Clr-b interactions to escape immune surveillance by wild-type but not Nkrp1b-/- NK cells. These data provide direct genetic evidence of a role for NKR-P1B in NK cell tolerance and MHC-I-independent missing-self recognition.