Trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2; also known as HER-2/neu), is indicated for the treatment of women with either early stage or metastatic HER2(+) breast cancer. It kills tumor cells by several mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Strategies that enhance the activity of ADCC effectors, including NK cells, may improve the efficacy of trastuzumab. Here, we have shown that upon encountering trastuzumab-coated, HER2-overexpressing breast cancer cells, human NK cells become activated and express the costimulatory receptor CD137. CD137 activation, which was dependent on NK cell expression of the FcγRIII receptor, occurred both in vitro and in the peripheral blood of women with HER2-expressing breast cancer after trastuzumab treatment. Stimulation of trastuzumab-activated human NK cells with an agonistic mAb specific for CD137 killed breast cancer cells (including an intrinsically trastuzumab-resistant cell line) more efficiently both in vitro and in vivo in xenotransplant models of human breast cancer, including one using a human primary breast tumor. The enhanced cytotoxicity was restricted to antibody-coated tumor cells. This sequential antibody strategy, combining a tumor-targeting antibody with a second antibody that activates the host innate immune system, may improve the therapeutic effects of antibodies against breast cancer and other HER2-expressing tumors.