Increased endometrial stromal cell (ESC) survival and migration is responsible for the development and progression of endometriosis. However, little is known about the mechanisms underlying ESC survival and migration, and limited therapeutic strategies that are able to reverse these abnormalities are available. The present study investigated the effects of zearalenone (ZEA) on ESC survival and migration, particularly focusing on mitochondrial fission and the c-Jun N-terminal kinase (JNK)/dynamin-related protein 1 (Drp1) pathway. The results revealed that ZEA induced ESC apoptosis in a dose-dependent manner. Furthermore, ZEA treatment triggered excessive mitochondrial fission resulting in structural and functional mitochondrial damage, leading to the collapse of the mitochondrial membrane potential and subsequent leakage of cytochrome c into the cytoplasm. This triggered the mitochondrial pathway of apoptosis. Additionally, ZEA-induced mitochondrial fission decreased ESC migration through F-actin/G-actin homeostasis dysregulation. ZEA also increased JNK phosphorylation and subsequently Drp1 phosphorylation at the serine 616 position, resulting in Drp1 activation. JNK/Drp1 pathway inhibition abolished the inhibitory effects of ZEA on ESC survival and migration. In summary, the present study demonstrated that ZEA reduced ESC survival and migration through the stimulation of mitochondrial fission by activation of the JNK/Drp1 pathway.