Deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), as induced by dexamethasone (DEX), is believed to play an important role in the onset of glucocorticoid-induced glaucoma (GIG). Abnormal ECM deposition is a consequence of mitochondrial dysfunction. We aimed to clarify how mitochondrial dysfunction leads to ECM deposition within the TM and to support the development of novel therapeutic strategies. In primary human TM cells (pHTMCs) and a dexamethasone acetate-induced murine model of GIG, glucocorticoid administration stimulated both mitochondrial fission and ECM deposition. Excessive mitochondrial fission leads to dysfunction and the overexpression of ECM proteins in pHTMCs. Notably, when pHTMCs were treated with the Drp1 inhibitor Mdivi-1 or with Drp1 siRNA, we observed a marked reduction in DEX-induced mitochondrial damage and ECM proteins in vitro. Furthermore, in C57BL/6J mice, treatment with Mdivi-1 mitigated mitochondrial damage and blocked ECM deposition within the TM. We then employed Ro3306 to inhibit the CDK1-mediated phosphorylation of Drp1 at Ser 616, which restored mitochondrial function and diminished DEX-induced ECM protein expression in pHTMCs. This study illuminates the pathogenic mechanism linking mitochondrial dysfunction to ECM deposition in GIG. Our innovative approach revealed that DEX stimulates mitochondrial fission via CDK1-mediated p-Drp1s616 overexpression, which drives ECM accumulation. It offered a novel therapeutic strategy for reducing ECM protein expression by inhibiting excessive mitochondrial fission and restoring mitochondrial function. By targeting the CDK1/Drp1-driven mitochondrial fission process, we can counteract DEX-induced ECM deposition in the TM both in vivo and in vitro.