An antagonistic peptide called HRAP that binds to the human HER2 molecule was designed by our computational method. In silico docking study demonstrated the specific interaction of HRAP with the dimerization domain in the HER2 molecule. Interestingly, HRAP inhibited proliferation of HER2-overexpressed human breast cancer cell lines. However, it had little cellular cytotoxicity (apoptosis inducibility). The cell proliferation inhibition was associated with the suppression of phosphorylation of PTEN and Akt. Thus, HRAP is the first HER2-binding small peptide antagonist rationally designed by a computer-aided SBDD method and is useful for the development of peptide mimetics to generate novel anti-breast cancer drugs.