The Arctic springtail, Megaphorura arctica, survives sub-zero temperatures in a dehydrated state via trehalose-dependent cryoprotective dehydration. Regulation of trehalose biosynthesis is complex; based in part on studies in yeast and fungi, its connection with oxidative stress caused by exposure of cells to oxidants, such as hydrogen peroxide (H2O2), or dehydration, is well documented. In this respect, we measured the amount of H2O2 and antioxidant enzyme activities (superoxide dismutases: copper, zinc—CuZnSOD and manganese containing–MnSOD, and catalase—CAT), as the regulatory components determining H2O2 concentrations, in Arctic springtails incubated at 5 °C (control) versus −2 °C (threshold temperature for trehalose biosynthesis). Because ecdysone also stimulates trehalose production in insects and regulates the expression of genes involved in redox homeostasis and antioxidant protection in Drosophila, we measured the levels of the active physiological form of ecdysone—20-hydroxyecdysone (20-HE). Significantly elevated H2O2 and 20-HE levels were observed in M. arctica incubated at −2 °C, supporting a link between ecdysone, H2O2, and trehalose levels during cryoprotective dehydration. CAT activity was found to be significantly lower in M. arctica incubated at −2 °C versus 5 °C, suggesting reduced H2O2 breakdown. Furthermore, measurement of the free radical composition in Arctic springtails incubated at 5 °C (controls) versus −2 °C by Electron Paramagnetic Resonance spectroscopy revealed melanin-derived free radicals at −2 °C, perhaps an additional source of H2O2. Our results suggest that H2O2 and ecdysone play important roles in the cryoprotective dehydration process in M. arctica, linked with the regulation of trehalose biosynthesis.