Tetraconazole, a systemic triazole fungicide, shows potential toxic effects in agriculture and human health. Therefore, its cytotoxic effects and accompanying mechanisms should be unraveled. S. pombe (ED666) was used in this study, as a unicellular biology and toxicology model. Cells were grown on standard media and all treatments were done at 30 C and shaking at 180 rpm 1-10 mg/L tetraconazole induced a dose-dependent cell death. Apoptosis was monitored by DAPI ve AO/EB staining. Excessive ROS production and mitochondrial impairment were shown by DCFDA/NBT assays and Rhodamine 123 staining, which were supported by increased expressions of superoxide dismutases and glutathione peroxidase. Involvement of one of the potential apoptotic genes, Cnx1, in apoptosis was shown by increased transcription whereas two other potential genes, Pca1 and Aif1, were not affected by tetraconazole treatment. In conclusion, tetraconazole-induced cytotoxicity and underlying mechanisms which were mediated via ROS damage and mitochondrial dysregulation (Cnx1-driven) were clarified in S. pombe.