Recent studies suggested that human CD56 bright CD16− NK cells may play a role in the regulation of the immune response. Since the mechanism (s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56 dim CD16+ and CD56 bright CD16− NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56 bright CD16− than in CD56 dim CD16+ NK cells. CD57 was mostly expressed by CD56 dim CD16+ NK cells. CD203a/PC-1 expression was restricted to CD56 bright CD16− NK cells. CD56 bright CD16− NK cells produce ADO and inhibit autologous CD4+ T cell proliferation. Such inhibition was 1) reverted pretreating CD56 bright CD16− NK cells with a CD38 inhibitor and 2) increased pretreating CD56 bright CD16− NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56 bright CD16− NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4+ T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56 bright CD16− NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56 bright CD16− NK cells act as “regulatory cells” through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases.