This study aims to determine the specificity of anti-human epidermal growth factor receptor antibody (anti-HER2) modified monomethoxy polyethylene glycol-chitosan (mPEG-CS) nanoparticles (anti-HER2/mPEG-CS NPs) in delivering small interfering RNA (siRNA) to the human epidermal growth factor receptor 2 (HER2) positive cancer cells. Physicochemical properties of the siRNA-loaded anti-HER2/mPEG-CS NPs (anti-HER2/mPEG-CS-siRNA NPs), including size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by particle size and zeta potential analyzer, and ultraviolet-visible spectrophotometer. MTT assay was used to study the in vitro cytotoxicity of the NPs. Fluorescent microscope and flow cytometer analysis results showed that anti-HER2/mPEG-CS-siRNA NPs had much efficient delivery of siRNA than the siRNA alone, Lipofectamine-siRNA complexes and mPEG-CS-siRNA NPs. These results demonstrated that anti-HER2/mPEG-CS-siRNA NPs had great potential applications as a targeted strategy for siRNA delivery.