Anti-HER2 antibody can induce antitumor responses, and can be used in delivering drugs to HER2-overexpressing cancer. Previously, we produced hyperthermia using magnetite nanoparticles that generate heat in an alternating magnetic field. In the present study, we constructed anti-HER2 immunoliposomes containing magnetite nanoparticles, which act as tumor-targeting vehicles, combining anti-HER2 antibody therapy with hyperthermia. The magnetite nanoparticle-loaded anti-HER2 immunoliposomes exerted HER2-mediated antiproliferative effects on SKBr3 breast cancer cells in vitro. Moreover, 60% of magnetite nanoparticles were incorporated into SKBr3, and the cells were then heated at 42.5 degrees C under an alternating magnetic field, resulting in strong cytotoxic effects. These results suggest that this novel therapeutic tool is applicable to treatment of HER2-overexpressing cancer.