PURPOSE: Silicone oil (SiO) is commonly employed as an intravitreal tamponade to manage complex retinal detachments associated with proliferative diabetic retinopathy, trauma, or severe myopia and to facilitate retinal reattachment. Nevertheless, SiO usage is linked to several complications, notably secondary glaucoma, which constitutes a significant proportion of adverse effects. This study investigated the impact of SiO on trabecular meshwork cells, given their pivotal role in regulating aqueous humor outflow. METHODS: Human trabecular meshwork cells (HTMCs) were co-cultured with SiO. The impact on proliferation, fibrosis-related markers, and ferroptosis levels on these cells was evaluated using 5-ethynyl-2'-deoxyuridine (EdU), western blot, and immunofluorescence assays. Further gene knockdown experiments with NOX4 and Smad3 were conducted to elucidate the underlying mechanisms of SiO-induced changes. RESULTS: SiO intervention inhibited HTMC proliferation, upregulated fibrosis-related markers, and elevated ferroptosis levels. Gene knockdown experiments revealed that SiO-induced ferroptosis and reactive oxygen species (ROS) increase were mediated through NOX4 upregulation and Smad3 activation. CONCLUSIONS: These findings highlight the significance of ferroptosis and the ROS/NOX4/Smad3 axis in the mechanism of SiO-induced intraocular pressure elevation. The insights gained from this study identify potential therapeutic targets to mitigate postoperative complications associated with SiO tamponade in ophthalmic surgery.