Matrine (MT), an active ingredient derived from Sophor flavescens Ait, is used as a therapeutic agent to treat liver disease and cancer. However, the serious toxic effects of MT, including nephrotoxicity, have limited its clinical application. Here, we explored the involvement of ferroptosis in MT-induced kidney injury and evaluated the potential efficacy and underlying mechanism of sodium selenite (SS) in attenuating MT-induced nephrotoxicity. We found that MT not only disrupts renal structure in mice but also induces the death of NRK-52E cells. Additionally, MT treatment resulted in significant elevations in ferrous iron, reactive oxygen species (ROS) and lipid peroxidation levels, accompanied by decreases in glutathione (GSH) and glutathione peroxidase (GPx) levels. SS effectively mitigated the alterations in ferroptosis-related indicators caused by MT and prevented MT-induced nephrotoxicity as effectively as Fer-1 in vivo and in vitro. SS also reversed the MT-induced reduction in GPX4, CTH and xCT protein levels. However, the glutathione peroxidase 4 (GPX4) inhibitor RSL3 and knockdown of GPX4, CTH, or xCT via siRNA abolished the protective effect of SS against MT-induced nephrotoxicity, indicating that SS exhibited antiferroptotic effects via the GSH-GPX4 antioxidant system. Overall, MT-induced ferroptosis triggers nephrotoxicity, and SS is a promising therapeutic drug for alleviating MT-induced renal injury by activating the GSH-GPX4 axis.