Objective To explore the regulatory effect and mechanism of calycosin on oxidative stress injury of SY5Y cells induced by H₂O₂ . Methods MTT assays were used to assess the effect of calycosin on the activity of SY5Y cells to screen for the appropriate drug concentration; The experiment included a PBS control group, H₂O₂(250 μmol / L) model group, and H₂O₂(250 μmol / L) + calycosin (0. 035 μmol / mL) intervention group. Kits were used to detect MDA content and SOD activity. Immunofluorescence staining was used to detect nuclear transfer of Nrf2 and expression of HO-1. Western blotting detected the levels of total Nrf2, nuclear Nrf2, NQO1, and HO-1 proteins. Results Compared with the PBS control group, the activity of SY5Y cells in the H₂O₂ group was reduced and calycosin improved H₂O₂ -induced cell damage, reduced the level of MDA, and increased the activity of SOD. Calycosin also activated Nrf2 and promote its translocation into the nucleus, which significantly upregulated the levels of total Nrf2, nuclear Nrf2, NQO1, and HO-1 after H₂O₂ injury. Conclusions Calycosin inhibited H₂O₂ -induced oxidative stress injury in SY5Y cells by activating Nrf2 / HO-1 signaling.