In this study, the potential toxicity of α-Fe2O3-NPs was investigated using a unicellular eukaryote model, Saccharomyces cerevisiae (S. cerevisiae). The results showed that cell viability and proliferation were significantly decreased (p < 0.01) following exposure to 100-600 mg L-1 for 24 h. The IC50 and LC50 values were 352 and 541 mg L-1, respectively. Toxic effects were attributed to α-Fe2O3-NPs rather than iron ions released from the NPs. α-Fe2O3-NPs were accumulated in the vacuole and cytoplasm, and the maximum accumulation (3.95 mg g-1) was reached at 12 h. About 48.6% of cells underwent late apoptosis/necrosis at 600 mg L-1, and the mitochondrial transmembrane potential was significantly decreased (p < 0.01) at 50-600 mg L-1. Biomarkers of oxidative stress [reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] and the expression of apoptosis-related genes (Yca1, Nma111, Nuc1 and SOD) were significantly changed after exposure. These combined results indicated that α-Fe2O3-NPs were rapidly internalized in S. cerevisiae, and the accumulated NPs induced cell apoptosis mediated by mitochondrial impairment and oxidative stress.