Background: Breast cancer treatment-failure is related to low compliance rates, high costs, and long-term toxicities. Thus, it is necessary to find less toxic and cheaper treatments. The epimereandrostene-3beta,17alpha-diol (α-AED) has been proven to inhibit breast tumor cell proliferation in vitro, being an ideal candidate to treat mammary tumors. The aim of this study was to evaluate the in situ effects of α-AED on tumor mammary growth. 4T1 tumors with 14 days of growth were injected with an equivalent dose to the IC50 (100 mM) and its double concentration (200 mM) of α-AED. Methods: The cell viability and cell proliferation of murine and human cancer cell lines after the treatment of α-AED were evaluated with sulforhodamine assay and bromodeoxyuridine incorporation, respectively. We also evaluated the size and tumor growth of the orthotopic tumor model after the treatment with α-AED. The tumor infiltration changes and cytokine determination into the tumor microenvironment were determined by flow cytometry and ELISA methods, respectively. The humoral response denoted by the production fo IgG anti-4T1 antibodies were also determined by ELISA methd. Results: Low and high concentrations of α-AED administrated intratumorally reduce the size and average tumor. α-AED also increased the percentages of NK, plasmatic and plasmablast cells in mice tumors injected with 100 mM of a-AED. Meanwhile, tumors injected with 200 mM of a-AED contained an elevated proportion of plasmablast and B cells. Of notice, VEGF levels in all a-AED-treated tumors were lower than the control and vehicle group. The tumor in situ response was reflected systemically by the increase of the anti-4T1 IgG in serum from a-AED-treated mice, but no other associated systemic changes were detected. The reduction in tumor size for to the local injection of a-AED is produced by the anti-proliferative effect of this hormone, that maybe associated to the VEGF reduction and metastasis. Conclusion: We observed an astonishing tumor size reduction and metastasis after the treatment with a-AED. The above suggest that a-AED can be used in clinical studies in order to prove its efficacy as an alternative of novel breast tumor treatment.