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In several carcinomas, the SET Domain Containing 3, Actin Histidine Methyltransferase (SETD3) is associated with oncogenesis. However, there is little knowledge about the role of SETD3 in the progression and prognosis of breast cancer. In this study, we first analyzed the prognostic value of SETD3 in breast cancer patients using the database of the public Kaplan-Meier plotter. Moreover, in vitro assays were performed to assess the role of SETD3 in the viability and capacity of invasion of human breast cancer cell lines. We observed that the high expression of SETD3 was associated with better relapse-free survival (RFS) of the whole collective of 3,951 patients, of Estrogen Receptor-positive, and of Luminal A-type breast cancer patients. However, in patients lacking expression of estrogen-, progesterone- and HER2-receptor, and those affected by a p53-mutation, SETD3 was associated with poor RFS. In vitro analysis showed that SETD3 siRNA depletion affects the viability of triple-negative cells as well as the cytoskeletal function and capacity of invasion of highly invasive MDA-MB-231 cells. Interestingly, SETD3 regulates the expression of other genes associated with cancer such as β-actin, FOXM1, FBXW7, Fascin, eNOS, and MMP-2. Our study suggests that SETD3 expression can act as a subtype-specific biomarker for breast cancer progression and prognosis. [ABSTRACT FROM AUTHOR]