PURPOSE: This study aims to explore how lipocalin 2 (LCN2) connects endoplasmic reticulum (ER) stress and inflammation in optic nerve injury (ONI) and identify potential therapeutic strategies. METHODS: An optic nerve crush (ONC) mouse model was used to investigate the role of ER stress and LCN2 in ONI. Immunofluorescence, quantitative PCR, and Western blot analyses were performed to assess ER stress markers, LCN2, inflammation-related genes, and retinal ganglion cell (RGC) survival, with or without treatment of 4PBA (an ER stress inhibitor) and TUN (an ER stress activator) in both the ONC model and BV2 cells. Lcn2 knockdown was achieved using small interfering RNA in BV2 cells and adeno-associated virus (AAV)-mediated gene silencing in vivo to explore underlying signaling pathways. RESULTS: ER stress markers (GRP78, ATF4, CHOP) and LCN2 expression were increased in ONC retinas, accompanied by microglial activation and RGC loss. Inhibition of ER stress using 4PBA effectively decreased LCN2 expression, attenuated microglial activation, and increased RGC survival post-ONC. Intravitreal injection of recombinant LCN2 induced a proinflammatory phenotype in microglia and exacerbated neurotoxicity. AAV-mediated Lcn2 silencing mitigated microglial activation, reduced neuroinflammation, and provided RGC neuroprotection, surpassing 4PBA treatment. In vitro studies further confirmed that Lcn2 knockdown significantly reduced the inflammatory response in BV2 cells by inhibiting NLRP3 inflammasome activation via the TLR4/NF-κB pathway. CONCLUSIONS: This study elucidates the critical role of LCN2 in linking ER stress and inflammation in ONI, offering a promising therapeutic target. AAV-mediated Lcn2 silencing outperforms broad ER stress inhibition, providing a novel strategy for treating optic nerve injuries.