Objective To investigate the protective effect of molecular hydrogen against hydrogen peroxide（H2O2）-induced oxidative injury and the correlated mechanism in primary cultures of rat spinal cord neurons. Methods Primary cultures of rat spinal cord neurons were cultured for 7 days, and then were randomly assigned to four groups with different treatments. (1) Normal medium (NM) control: continued to culture with normal medium. (2) Hydrogen-rich medium（HM）: continued to culture with hydrogen-riched medium. (3) NM + H2O2: cultured with NM, and treated with 100 μM H2O2 and 15 μM ferrous chloride (FeCl2) after pretreatment of HM for 2 h. (4) HM + H2O2: cultured with HM containing 100 μM H2O2 and 15 μM FeCl2 after pretreatment of HM for 2 h. The respective media were changed every 6 h. Treatments were carried out for 12 h, and then the cells were collected for assays of reactive oxygen species (ROS), hydroxyl radical (HO•), apoptosis, and the expression of Glycogen synthase kinase-3β (GSK-3β) and p-GSK-3β. Results Compared with NM, HM could reduce the H2O2-induced intracellular production of ROS and HO• in purified neurons (P ＜ 0.01), and the number of apoptotic neurons was significantly decreased (P ＜ 0.01) with the downregulation of Caspase-3. In addition, HM also promoted the phosphorylation of GSK-3β (P ＜ 0.01). Conclusion Molecular hydrogen possessed the protective effect against H2O2-induced oxidative injury in primary cultured neurons. The mechanisms were related to reducing the intracellular production of ROS and HO•, inhibiting the activation of apoptosis in neurons and increasing the phosphorylation of GSK-3β to promote growth of neurons.