CD96 has recently been shown to be a potent immune checkpoint molecule in mice, but a similar role in humans is not known. In this study, we provide a detailed map of CD96 expression across human lymphocyte lineages, the kinetics of CD96 regulation on T‐cell activation and co‐expression with other conventional and emerging immune checkpoint molecules. We show that CD96 is predominantly expressed by T cells and has a unique lymphocyte expression profile. CD96^high T cells exhibited distinct effector functions on activation. Of note, CD96 expression was highly correlated with T‐cell markers in primary and metastatic human tumors and was elevated on antigen‐experienced T cells and tumor‐infiltrating lymphocytes. Collectively, these data demonstrate that CD96 may be a promising immune checkpoint to enhance T‐cell function against human cancer and infectious disease.