Mitochondrial transcription termination factor 3 (MTERF3) is a negative regulator of mitochondrial transcription. It is a modular factor involves in mitochondrial ribosome biogenesis and protein synthesis. However, the association between MTERF3 and breast cancers remains largely unknown. The aim of this study was to investigate the expression of MTERF3 in breast carcinoma and to analyze its clinicopathological significance, and to examine the potential prognostic value of MTERF3 in breast cancer.The protein expression levels of MTERF3 in MCF7 (Luminal A), BT-474 (Luminal B), SKBR3 (HER2 overexpression), MDA-MB-468 (Basal like) and MCF10A cell lines were detected by Western blotting. Immunohistochemistry (IHC), Western blotting, and semiquantitative RT-PCR were performed to analyze the protein and mRNA expression levels of MTERF3 in 58 breast cancer tissues and 58 noncancerous breast tissues. The MTERF3 expression data and clinical information from breast cancer patients were downloaded from the TCGA dataset by using the R3.6.1 software. Then the relationship between the expression level of MTERF3 and clinicopathological characteristics and the prognostic value was analyzed. A Cox regression model was performed for the multivariate analysis of the factors that affected the prognosis of breast cancer. The association between the expression levels of MTERF3 and other mitochondrial regulatory genes was analyzed with GEPIA.MTERF3 is upregulated in breast cancer cell lines compared to noncancerous breast cell line. The IHC results showed that the MTERF3 protein was located in the cytoplasm, and the rate of positive expression in breast cancer tissue was significantly upregulated compared with the adjacent normal tissue. The mRNA and protein expression levels of MTERF3 in breast cancer tissues were significantly higher than that in breast tissues. Moreover, the expression of MTERF3 was significantly correlated with ER status, PR status, breast cancer molecular typing, cancer type, histological diagnosis and primary site (P<0.05). Further analysis showed MTERF3 expression was not related to prognosis. Multivariate Cox regression analysis showed that age, metastasis status and tumor type were independent prognostic factors for breast cancer patients. The expression levels of MTERF3 were positively correlated with the TFAM, TFB1M, TFB2M, MTERF1, TEFM and MFN1 genes but negatively correlated with the MTERF4 and PINK1 genes. In addition, the expression levels of MTERF3 were not correlated with the MTERF2 gene.MTERF3 was significantly upregulated in breast cancer cells and tissues compared with noncancerous cells and tissues. Moreover, the expression level of MTERF3 was correlated with ER status, PR status, breast cancer molecular typing, cancer type, histological diagnosis and primary site. These findings suggested that the upregulation of MTERF3 may be used as a diagnostic and therapeutic target in breast carcinoma.2020 Translational Cancer Research. All rights reserved.