H9N2 influenza virus, a prevalent influenza A virus, causes acute lung injury through mitochondrial damage associated with oxidative stress. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+ permeable non-selective cation channel that can trigger oxidative stress via Ca2+ overload. Excessive ROS generation leads to mitochondrial dysfunction and lipid peroxides accumulation, contributing to ferroptosis. However, it remains unclear whether H9N2 virus infection can trigger ferroptosis in mouse lungs and its relationship with TRPM2. Therefore, this study investigates the protective effect of TRPM2 knockdown against lung injury infected by H9N2 virus and explores its potential molecular mechanisms, with a particular focus on its association with ferroptosis. In vitro, we infected mouse pulmonary microvascular endothelial cells (PMVECs) with H9N2 virus, or/and transfected them with siTRPM2 at 80 nM. Our findings revealed that TRPM2 knockdown significantly reduced Ca2+ overload and ROS generation, and upregulated the mRNA and protein expression levels of catalase (CAT), superoxide dismutase 1 (SOD1), and heme oxygenase-1 (HO-1). This intervention also alleviated mitochondrial damage and maintained mitochondrial dynamics balance. H9N2 virus infection disrupted the Glutathione/Glutathione oxidized (GSH/GSSG) system and increased lipid peroxidation-related factors (Lysophosphatidylcholine acyltransferase 3 [LPCAT3] and Acyl-CoA synthetase long chain family member 4 [ACSL4]), which were mitigated by TRPM2 knockdown. Additionally, TRPM2 ablation reduced Fe2+ intensity and the expression levels of iron metabolism-related factors (Transferrin [TF] and Transferrin receptor [TFR]). In conclusion, TRPM2 knockdown inhibited H9N2 virus-induced ferroptosis by mitigating Ca2+ overload, oxidative stress, mitochondrial dysfunction, GSH/GSSG system imbalance, lipid peroxidation, and iron metabolism imbalance.