Many insects in temperate regions overwinter in diapause, during which they are cold hardy. In these insects, one of the metabolic adaptations to the unfavorable environmental conditions is the synthesis of cryoprotectants/anhydroprotectants. The aim of this study was to investigate the connection between the antioxidative system and synthesis of cryoprotectants (mainly glycerol) in diapausing larvae of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae). At two periods during diapause (November and February), in three groups of insects (kept under field conditions; -12°C for two weeks; 8°C for two weeks), the activity of key enzymes of the antioxidative system and oxidative part of the hexose monophosphate shunt were measured: superoxide dismutase, catalase, non selenium glutathione peroxidase, glutathione S-transferase, glutathione reductase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, as well that of the antioxidative components: total glutathione and ascorbate, and dehydroascorbate reductase. There was a higher activity of antioxidative enzymes at the beginning of the diapause period (November) compared to late diapause (February), while glutathione and ascorbate were higher in February. Similarly, there was a lower activity of the hexose monophosphate shunt enzymes in February. Exposure of larvae to -12°C resulted in an elevation of hexose monophosphate shunt enzyme activity, especially in November. This was accompanied by a significant increase in glycerol content in February. Changes in ascorbate levels and dehydroascorbate reductase activity in both experimental groups (-12°C and 8°C) suggest a connection between the antioxidative system, metabolism during diapause and cold hardiness. Our results support the notion that antioxidative defense in larvae of Ostrinia nubilalis is closely connected with metabolic changes characteristic of diapause, mechanisms of cold hardiness involved in diapause and the maintenance of a stable redox state.