Resistance to doxorubicin (DOX) remains a big challenge to breast cancer treatment especially for triple negative breast cancer (TNBC). Our previous study revealed that the antioxidant system plays an important role in conferring metastasis derived DOX resistance. In this study, we used two-dimensional difference gel electrophoresis (2D-DIGE) proteomics to compare the expression profiles of two generations of TNBC cell lines which have increased metastatic ability in nude mice and exhibited resistance to DOX. Through careful analyses, one antioxidant protein: glucose-6-phosphate dehydrogenase (G6PD) was identified with 3.2-fold higher level in metastatic/DOX-resistant 231-M1 than its parental 231-C3 cells. Analyses of clinical data showed that TNBC patients with higher G6PD levels exhibited lower overall survival than patients with lower G6PD level. Reducing G6PD expression by siRNA or inhibiting its activity with dehydroepiandrosterone (DHEA) significantly increased DOX's cytotoxicity in both cell lines. Importantly, inhibiting G6PD's activity with DHEA dramatically increased the apoptotic rate of 1.25 µM DOX from 2% to 54%. Our results suggest that high level of G6PD can help TNBC to resist DOX-induced oxidative stress. Thus, inhibiting G6PD shall be a good strategy to treat DOX-resistant TNBC.