Modulation of the immune response using immunoglobulin fusion proteins has shown great promise for clinical immunotherapy of autoimmune diseases. Alefacept is an immunoglobulin fusion protein composed of the first extracellular domain of human LFA‐3 fused to the hinge, C_H2 and C_H3 domains of human IgG₁. Alefacept has previously been reported to inhibit T cell proliferation. Here, we analyzed the effects of alefacept on lymphocytes in vitro and characterized the role of autologous NK cells in its mechanism of action. Alefacept, but not a C_H2 binding mutant of Alefacept, inhibited CD3‐induced T cell proliferation only in the presence of live NK cells, consistent with an important role for FcγR engagement. Alefacept caused preferential depletion of CD69⁺CD45R0⁺CD25⁺ T cell subsets. Cytotoxicity assays revealed that alefacept, but not the C_H2 binding mutant, induced NK cell‐mediated death of activated T cells and sorting into CD45R0⁺ and CD45RA⁺ subpopulations showed that lymphocyte deletion occurred preferentially in the CD45R0⁺ subset. Activated CD45R0⁺ cells expressed higher levels of CD2 than CD45R0^– cells, providing a possible explanation for the selective targeting of this subset. Our results suggest that selective targeting of CD45R0⁺ T cells by NK cells represents a potential therapeutic mechanism of action of alefacept.