Given the great potential of superparamagnetic iron oxide nanoparticles (SPIONs) in the application of biomedical and water treatment fields, it is of vital significance to evaluate their toxicity and explore the underlying mechanism. Silica-coated SPIONs (SPIONs@SiO2) might generate oxidative stress and cytotoxicity by interfering antioxidant enzyme. This study investigated the response of superoxide dismutase (SOD) under SPIONs@SiO2-induced oxidative stress using a strategy integrating cellular and molecular methods. Results showed that intracellular SOD activity exhibited hormetic effects in response to SPIONs@SiO2-induced structural changes of SOD molecule and oxidative stress, which subsequently generated lipid oxidation and cell viability loss in the mouse primary hepatocytes. The structural changes and increasing activity of SOD molecule were resulted from the direct interaction with SPIONs@SiO2. Then, multi-spectroscopy, zeta potential measurements and isothermal titration calorimetry were used to study the interaction of SPIONs@SiO2 with SOD and the underlying mechanism. SOD interacts with SPIONs@SiO2 through electrostatic and hydrophobic forces, enhancing the hydrophobicity around Tyr 108. The enthalpy and entropy change of the interaction are 1.03 × 105 J.mol−1 and 474.6 J.mol−1.K−1, respectively, indicating the dominant driving forces are hydrophobic forces. The interaction led to the loosen of peptide chains and the reduction of aggregation state. And the α-helix content of SOD decreased to 13 % in the presence of SPIONs@SiO2.