PURPOSE: Fungal keratitis (FK) is a challenging and sight-threatening corneal disease caused by fungal infections. Although long noncoding RNAs (lncRNAs) have been explored in various infectious diseases, their specific roles in FK remain largely unexplored. METHODS: A mouse model of FK was created by infecting corneal stromal cells with Fusarium solani. High-throughput lncRNA expression profiling was conducted on FK-affected corneal tissues to identify differentially expressed lncRNAs. Reverse transcription quantitative PCR (RT-qPCR) was used to validate the results. A competing endogenous RNA (ceRNA) network was constructed. Additionally, a specific antisense oligonucleotide (ASO) targeting lncRNA ENSMUST00000226838/Osr2 (Osr2) was developed for therapeutic evaluation. Inflammatory markers IL-1β, IL-6, and TNF-α were measured, and corneal inflammation was assessed through histological analysis and slit-lamp examination. Fluorescent in situ hybridization (FISH) was used to confirm Osr2 localization, whereas a dual-luciferase reporter assay verified interactions between Osr2 and miR-30a-3p. RESULTS: We identified 1143 differentially expressed lncRNAs in FK, with 701 upregulated and 442 downregulated. The ceRNA network analysis indicated that lncRNA Osr2 regulates Xcr1 expression through miR-30a-3p. Treatment with ASO-Osr2 significantly reduced corneal inflammation, and FISH confirmed lncRNA Osr2 distribution in both the nucleus and cytoplasm. Dual-luciferase assays demonstrated the interaction between Osr2 and miR-30a-3p, highlighting their potential roles in the progression of FK. CONCLUSIONS: This study outlined the lncRNA expression profile in FK and established a ceRNA regulatory network, identifying lncRNA Osr2 as a crucial modulator of FK pathogenesis through its interaction with miR-30a-3p. These findings highlighted lncRNA Osr2 as a promising therapeutic target for the treatment of FK.