Breast cancer (BC) is the most common cancer and the second leading cause of cancer related deaths among women in the United States. Despite important advances in treatment, breast cancer remains incurable due to the emergence of alternative pathways adopted by cancer cells to overcome the effects of anti-cancer therapy. This molecular heterogeneity in the cellular response to anti-cancer therapy challenges the development of effective therapeutic strategies. To overcome this issue, a rational approach would be to concomitantly target clinically relevant cellular abnormalities with combination therapy or to use a potent multi-targeted agent. Autophagy is a critical physiological process of cancer cell survival in the presence of chemotherapeutic stress and is an important cause of resistance to anti-cancer therapy. Drugs that inhibit autophagy e.g. chloroquine-(CHL) deprive the cells of this protective mechanism and therefore are promising agents in combination chemotherapy of breast cancer. All-trans retinoic acid (ATRA), a potent differentiation inducing agent, has been tested as anticancer agent for a number of cancers including breast cancer, but its rapid metabolism to inactive metabolites is a major problem in its clinical use. VN/12-1, a novel retinoic acid metabolism blocking agent (RAMBA) developed in our laboratory, showed improved anti-proliferative activity over ATRA against estrogen receptor α (ER-α) positive and negative breast cancer cell lines. Human epidermal growth factor receptor-2 (Her-2) is a critical protein involved cell proliferation and metastasis. Her-2 has been involved in increasing the frequency of tumor stem cells and it has also been implicated in the development of drug resistance. Hence, for our current studies, we primarily focused on a cell line which is deficient in ER-α and which overexpresses Her-2 (SKBR-3 cells). We showed here that VN/12-1 inhibits G1-S phase cell cycle transition. However, short treatment of VN/12-1 induced autophagy in these cells and protected cells from apoptosis. VN/12-1-induced autophagy was preceded by ER stress (ERS). We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of Beclin-1 enhanced cell death induced by VN/12-1 in SKBR-3 cells. The combination of VN/12-1with autophagy inhibitor - CHL resulted in synergistic effects in that it enhanced apoptosis in vitro and in vivo which was caspase dependent. Importantly, VN/12-1 (5 mg/kg twice weekly) and VN/12-1 (5 mg/kg twice weekly) + chloroquine (50 mg/kg twice weekly) significantly suppressed established SKBR-3 tumor growth by 81.4% (p < 0.001 vs. control) and 96.2% (p < 0.001 vs control), respectively without incurring overt signs of toxicity or significant loss of body weight. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of RAMBAs in the treatment of breast cancer. It is hoped that this combination therapy would solve the problem of the development of acquired resistance due to autophagy that typically follows the chemotherapeutic treatment of breast cancer. We envision further advanced preclinical development of this combination therapy as potential therapy for breast cancer.