Breast carcinoma is a major health issue for millions of women. Current therapies have serious side effects, and are only partially effective in patients with metastatic tumors. Thus, the need for novel and less toxic therapies is urgent. Moreover, hormonal and antibody therapies effective in other subtypes are not effective in Triple Negative Breast Cancer (TNBC). Immunotherapeutic strategies directed against specific tumor-associated antigens (TAAs) and mediated by specific cytotoxic T lymphocytes (CTL) have been largely underexplored in this disease. Cancer-testis antigens (CTA) are a group of TAAs displaying the ideal characteristics of promising vaccine targets, i.e. strong immunogenicity and cancer specificity. The CTA, Sperm Protein 17 (SP17), has been found to be aberrantly expressed in different neoplasms, including ovarian and esophageal cancers, nervous system tumors and multiple myeloma, and has been suggested as a candidate target for immunotherapy. Here, we evaluated SP17 expression levels in breast cancer cell lines, invasive ductal breast carcinoma, including patients with TNBC, and adjacent non-neoplastic breast tissue, and determined whether SP17 was capable of generating SP17-specific cytotoxic T lymphocytes in vitro. We showed that SP17 is expressed in breast cancer cell lines and primary breast tumors and importantly in TNBC subtype, but not in adjacent non-tumoral breast tissue or unaffected tissues, except in male germinal cells. Furthermore, we detected specific anti-SP17 antibodies in patients' sera and we generated SP17-specific, HLA class I-restricted, cytotoxic T lymphocytes capable of efficiently killing breast cancer cells.