Pulmonary arterial hypertension (PAH) is characterized by pulmonary artery smooth muscle cell (PASMC) dysfunction. However, the underlying mechanisms of PASMC dysfunction remain largely unknown. Here, we show that mitochondrial fragmentation contributes to PASMC dysfunction through enhancement of endoplasmic reticulum (ER) stress. PASMC dysfunction accompanied by mitochondrial fragmentation and ER stress was observed in the pulmonary arteries of hypoxia-induced rats with PAH, as well as isolated PASMCs under hypoxia. Treatment with Mdivi-1 inhibited mitochondrial fragmentation and ER stress and improved PASMC function in isolated PASMCs under hypoxia, while Drp1 overexpression increased mitochondrial fragmentation and ER stress, impairing PASMC function in isolated PASMCs under normoxia. However, inhibition of ER stress using ER stress inhibitors showed a negligible effect on mitochondrial morphology but improved PASMC function during hypoxia. Additionally, we found that mitochondrial fragmentation-promoted ER stress was dependent on mitochondrial reactive oxygen species. Furthermore, inhibition of mitochondrial fragmentation using Mdivi-1 attenuated mitochondrial fragmentation and ER stress in hypoxic PASMCs and improved the pulmonary artery smooth muscle function in hypoxic rats. These results suggest that hypoxia induces pulmonary artery smooth muscle dysfunction through mitochondrial fragmentation-mediated ER stress and that mitochondrial morphology is a potential target for treatment of hypoxia-induced pulmonary artery smooth muscle dysfunction.