BACKGROUND: More than half of patients with neovascular age-related macular degeneration (nAMD) develop subretinal fibrosis, regardless of anti-vascular endothelial growth factor (VEGF) therapy. No treatment exists for subretinal fibrosis, as its pathophysiology remains elusive. Granzyme B (GzmB) is a serine protease elevated in human eyes with nAMD and contributes to choroidal neovascularization (CNV). Although GzmB is involved in dermal and cardiac fibrosis, its role in subretinal fibrosis has yet to be elucidated. METHODS: Using the two-stage laser-induced mouse model of subretinal fibrosis, fibrotic lesions were induced in younger (3–6 months old) and older (7–14 months old) C57BL/6J and GzmB deficient mice. Seven days after the second laser, in vivo imaging of fibrotic lesions was performed using custom-built polarization diversity-optical coherence tomography (PD-OCT) system. Eyes were collected and used for either retina wholemounts or cross-sections. Wholemounts were immunostained to assess pathological features of fibrosis and determine the size of fibrotic lesions and mast cell counts within fibrotic lesions. Cross-sections were processed to quantify the levels of GzmB substrates within fibrotic lesions, namely pro-fibrotic thrombospondin-1 (TSP-1) and anti-fibrotic decorin (DCN), the extent of macrophage-to-myofibroblast transition (MMT), activation of astrocytes/Müller cells and finally photoreceptor cell death. ARPE-19 wound healing assay was performed to study the direct role of GzmB in RPE wound healing in vitro. GzmB-mediated transcriptional changes in ARPE-19 were determined by performing bulk RNA sequencing. Immunocytochemistry was performed to assess epithelial-mesenchymal transition (EMT). RESULTS: GzmB deficiency resulted in smaller fibrotic lesions in older mice and was associated with decreased levels of TSP-1 and increased levels of DCN within fibrotic lesions. It also led to increased MMT but reduced mast cell counts within fibrotic lesions, which was correlated with reduced photoreceptor cell death. In ARPE-19, exogenous application of GzmB impaired wound closure and promoted partial EMT by selectively modulating genes involved in transforming growth factor-β signaling, EMT, inflammation, angiogenesis and cell-cell interaction. CONCLUSIONS: Our study reveals that extracellular GzmB is a key contributor to subretinal fibrosis in nAMD, modulating inflammation, EMT and photoreceptor degeneration. These findings suggest GzmB as a promising therapeutic target for mitigating the development of subretinal fibrosis in nAMD.