This study investigates the non-estrogenic mode of zearalenone (ZEA) toxicity in a novel aspect via accumulation of reactive oxygen species (ROS) and the regulation of the activities of antioxidant enzymes in the Schizosaccharomyces pombe in acute toxicity tests. In comparison with the control, 500 μM ZEA treatment caused 66% decrease in the concentration of glutathione (GSH), which was a consequence, in the absence of ZEA-GSH interaction, of the GSH-consuming processes of the antioxidant system; this depletion of GSH initiated a 1.8- and 2.0-fold accumulation of the superoxide anion and hydrogen peroxide, but did not increase the concentration of the hydroxyl radical; ROS-induced adaptation processes via activation of the Pap1 transcription factor resulted in significantly increased activities of superoxide dismutases, catalase, glutathione reductase and glutathione S-transferase, and decreased activities of glutathione peroxidase and glucose-6-phosphate dehydrogenase. This treatment altered the sterol composition of the cells by inducing decreased concentrations of ergosterol, squalene and 24-methylene-24,25-hydrolanosterol, and also elevated the number of fragmented nuclei. Cells strived to correct the unbalanced redox state by regulation of the antioxidant system, but this was not enough to defend the cells from the disturbed sterol composition, the cell cycle arrest, and the fragmentation of nuclei. 