Hemorrhagic stroke is associated with high morbidity and mortality. Hemin is a decomposition product of hemoglobin that is related to neuronal apoptosis after hemorrhage, although the molecular basis for this association remains unclear. To address this issue, the present study investigated hemin-induced changes in the apoptotic index and mitochondrial ultrastructure in SH-SY5Y cells. Cell viability was evaluated using Cell Counting Kit-8 and by terminal transferase dUTP nick-end labeling, western blotting, and flow cytometry. Changes in mitochondrial ultrastructure were examined by super-resolution three-dimensional structured illumination microscopy. We found that cleaved-caspase-3 expression and the number of apoptotic cells increased in a time-dependent manner upon hemin treatment, which was associated with mitochondrial fragmentation. Our data suggest that hemin induces apoptosis and mitochondrial fission in neuronal cells. Thus, therapeutic strategies that target hemin could mitigate the damage caused by hemorrhagic stroke.