NK cells express several tetraspanin proteins, which differentially modulate NK cell activities. The tetraspanin CD53 is expressed by all resting NK cells and was previously shown to decrease NK cell cytotoxicity upon ligation. Here, we show that CD53 ligation reduced degranulation of rat NK cells in response to tumour target cells, evoked redirected inhibition of killing of Fc-bearing targets, and reduced the IFN-γ response induced by plate-bound antibodies towards several activating NK cell receptors (Ly49s3, NKR-P1A, and NKp46). CD53 induced activation of the β2 integrin LFA-1, which was further enhanced upon co-stimulation with activating NK cell receptors. Concordant with a role for CD53 in increasing NK cell adhesiveness, CD53 ligation induced a strong homotypic adhesion between NK cells. Further, the proliferative capacity of NK cells to a suboptimal dose of IL-2 was enhanced by CD53 ligation. Taken together, these data suggest that CD53 may shift NK cell responses from effector functions towards a proliferation phase.