HER4 isoforms have oncogenic or tumor suppressor functions depending on their susceptibility to proteolytic cleavage and HER4 Intracellular Domain (4ICD) translocation. Here, we report that the NRG1 tumor suppressor mechanism through the HER4 JMa/CYT1 isoform can be mimicked by the agonist anti-HER4 antibody C6. NRG1 induced cleavage of poly(ADP-ribose) polymerase (PARP) and sub-G1 DNA fragmentation, and also reduced the metabolic activity of HER3-negative/HER4-positive cervical (C-33A) and ovarian (COV318) cancer cells. This effect was confirmed in HER4 JMa/CYT1-, but not JMa/CYT2-transfected BT549 triple-negative breast cancer cells. NRG1 favored 4ICD cleavage and retention in mitochondria in JMa/CYT1-transfected BT549 cells, leading to Reactive Oxygen Species (ROS) production through mitochondrial depolarization. Similarly, the anti-HER4 antibody C6, which binds to a conformational epitope located on aa 575-592 and 605-620 of HER4 domain IV, induced 4ICD cleavage and retention in mitochondria, and mimicked NRG1-mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization in cancer cells. In vivo, C6 reduced growth of COV434 and HCC187 tumor cell xenografts in nude mice. Biasing 4ICD trafficking to mitochondria with anti-HER4 antibodies to mimic NRG1 suppressor functions could be an alternative anti-cancer strategy.