Numerous reports find that Cd induces formation of reactive oxygen species (ROS) in plants. However, a general ROS pool is usually studied, without distinction of their production site. In the present study, we applied a mitochondria-specific antioxidant, MitoTEMPO, to elucidate the role of mitochondria-derived ROS in the response of soybean seedlings to short-term (48 h) Cd stress. The obtained results showed that Cd caused a reduction in root length and fresh weight and increase in the level of superoxide anion, hydrogen peroxide, markers of lipid peroxidation (thiobarbituric reactive substances, TBARS) and markers of RNA oxidation (8-hydroxyguanosine, 8-OHG) in seedling roots. Application of MitoTEMPO affected Cd uptake in a dose-dependent manner and diminished the Cd-dependent induction of superoxide anion and lipid peroxidation.