PURPOSE: Although the mechanisms underlying herpes simplex virus type-1 (HSV-1) ocular infection have been extensively studied, the role of host microRNAs (miRNAs) in the pathobiology of herpetic keratitis (HK) is not well understood. The aim of this study was to identify endogenous miRNA regulators involved in the progression of HSV-1 ocular infection. METHODS: C57BL/6 mice were infected with HSV-1 strain McKrae following epithelial debridement, and corneal miRNA profiles were analyzed at various time points using miRNA sequencing (miRNA-seq). The miRNA expression was measured at 2, 4, 6, and 10 days post-infection. Ingenuity Pathway Analysis (IPA) was used to identify immune pathways potentially targeted by differentially expressed miRNAs. The role of selected miRNAs in viral entry and replication was assessed by overexpression in murine embryonic fibroblasts (MEFs) and human corneal epithelial cells (HCEs). RESULTS: A total of 32 miRNAs at 2 days post-infection, 21 miRNAs at 4 days post-infection, 140 miRNAs at 6 days post-infection, and 27 miRNAs at 10 days post-infection showed significant changes in expression. IPA revealed that differentially expressed miRNAs targeted several immune pathways, including TLR and interferon signaling. Notably, mmu-miR-184-3p and mmu-let-7d-5p were upregulated, whereas mmu-miR-329-3p was down-regulated during infection. Functional assays demonstrated that overexpression of miR-329, but not miR-184-3p or miR-let-7d-5p, increased HSV-1 viral entry and replication in a dose-dependent manner. In contrast, miR-329 inhibition reversed these effects, suggesting its role as a pro-viral miRNA. Increased plaque formation and viral gB expression further confirmed miR-329's pro-viral role. CONCLUSIONS: Our findings suggest that miR-329 functions as a pro-viral miRNA by disrupting TLR9 signaling, thus facilitating HSV-1 replication. Inhibition of miR-329 enhances TLR9-mediated antiviral responses, highlighting the potential of targeting host miRNAs as a novel therapeutic strategy for managing viral keratitis.