The low affinity receptor for IgG, FcγRIIIA, CD16, is the prototype of NK activating receptors and is responsible for the Antibody Dependent Cellular Cytotoxicity (ADCC). It is well recognised that the clinical efficacy of a number of antibody-based therapeutic approaches largely relies on the ability to induce ADCC. Here we analysed the dynamics of CD16 expression in primary human NK cells during cytolytic interactions with opsonized targets and the impact of receptor trafficking on NK cytotoxic potential. The chimeric Rituximab and the human Ofatumumab anti-CD20 therapeutic monoclonal antibodies were used to opsonize human lymphoblastoid CD20-positive cell lines. Our findings demonstrate that the interaction with opsonized target cells induces a marked CD16 internalization associated with a reduced ADCC function in the absence receptor recycling. Interestingly, in opsonised target-activated NK cells we observed a major impairment of the spontaneous cytotoxic response against a panel of sensitive targets. Such CD16-dependent NK cell hyporesponsiveness was largely independent from the exhaustion of cytolytic mediators or from the down-modulation of activation receptor surface expression. Further, exploring the ability of individual receptor/ligand interaction to induce the cytotoxic response, we observed that CD16-induced cross-tolerance regards different activating receptors including NKG2D, DNAM-1 and to a lesser extent NKp46 and 2B4. Our data support the notion that stimulation via CD16 may lead to NK cell hyporesponsiveness to triggering via other unrelated signalling pathways.