SignificanceCD226 is an important activating receptor involved in mediating natural killer (NK) cell responses against tumors, but how CD226 exerts control over NK cell function is not fully understood. CD226 belongs to the poliovirus receptor (PVR)-nectin family that includes TIGIT and CD96, with TIGIT garnering much attention as a key checkpoint in T cell and NK cell antitumor responses and as an immunotherapy target. Thus, it is imperative to determine how CD226 counteracts the actions of TIGIT and CD96 with which it competes for binding to its ligands such as CD155 (PVR). We demonstrate that CD226 engagement of CD155 is required for phosphorylation of transcription factor FOXO1, resulting in inactivation of its negative regulatory control over NK cell effector function.