Mitochondria play a pivotal role in regulating the viability of cancer cells. The role of lapatinib in cancer is poorly understood. This study investigated the downstream response to mitochondrial fragmentation using lapatinib to activate mitochondria. In this study cell viability was measured by MTT and LDH leakage assays. Apoptosis was determined by enzyme-linked immunosorbent assay (ELISA) and Annexin V/PI staining. Mitochondrial fragmentation was measured by qPCR and protein expression was detected by Western blot. Results showed that lapatinib increased ovarian cancer cell apoptosis in SKOV-3 cells via triggering mitochondrial fragmentation. Subsequently, mitochondrial fragmentation increased ROS production and facilitated HtrA2/Omi liberation from the mitochondria into the cytoplasm which inturn activated caspase-dependent cell apoptosis. The present results identified the mROS-HtrA2/Omi axis as a novel signaling pathway that is activated by mitochondrial fragmentation and promotes ovarian cancer cell apoptosis with lapatinib treatment.