Aurora A has multiple key functions in tumor initiation and progression and is overexpressed in many cancers. Several ongoing clinical trials are assessing the unique therapeutic potential of Aurora-based targeted therapy, but several severe adverse events such as hematopoietic toxicity have been observed in the early-phase clinical trials because Aurora A is also involved in normal cells proliferation process. The strategy to develop tumor-specific inhibition of this target may be an alteration for the treatment of Aurora A overexpression tumors. In this study, we developed a novel tumor-specific RNA interference adenovirus system targeting Aurora A by using stathmin promoter and investigated the effects of it on the proliferation, apoptosis and chemotherapy sensitivity in human breast carcinoma cells both in vitro and in vivo. The results showed that treatment of human breast carcinoma cells (SK-BR-3 and MDA-MB-231) by Aurora A short hairpin RNA (shRNA) driven by stathmin gene promoter not only inhibited the cells proliferation, but also enhanced the chemosensitivity to paclitaxel via downregulation of Aurora A mRNA and protein expression, which further decreased the phosphatidylinositol 3 kinase/Akt and p-BRCA1 protein expression. Furthermore, there were no obvious phenotypes changes observed in normally differentiated epithelial cells of MCF210. Therefore, stathmin promoter-driving Aurora A shRNA adenoviral system may have potential use, with targeted tumor gene silencing effect and as adjuvant tumor-specific therapy method, in the treatment of human breast carcinomas.