The trabecular meshwork (TM) plays an essential role in the circulation of aqueous humor by sensing mechanical stretch. The balance between the outflow and inflow of aqueous humor is critical in regulating intraocular pressure (IOP). A dysfunctional TM leads to resistance to the outflow of aqueous humor, resulting in an elevated IOP, a major risk factor for glaucoma. It is widely accepted that mutant myocilin (MYOC) can cause damage to the TM. However, few studies have investigated how TM cells carrying mutant MYOC respond to cyclic mechanical stretch (CMS) and whether these cells are more sensitive to CMS under this genetic background. In this study, we applied mechanical stretch to TM cells using the Flexcell system to mimic physiological stress. In addition, we performed genome-wide transcriptome analysis and oxidized lipidomics to systematically compare the gene expression and oxylipin profiles of non-stretched control human primary TM cells, human primary TM cells under CMS (TM-CMS), and human primary TM cells overexpressing MYOCS341P under CMS (S341P-CMS). We found that TM cells that overexpressed MYOCS341P were more sensitive to mechanical stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that downregulated genes were most enriched in oxidative phosphorylation, indicating mitochondria dysfunction and the likelihood of oxidative stress. Oxidized lipidomics analysis revealed significant changes in oxylipin profiles between the S341P-CMS and TM-CMS groups. Through further genome-wide transcriptomic analysis, we identified several genes that may be involved in the sensitivity of TM cells that overexpressed MYOCS341P to mechanical stress, including SARM1, AHNAK2, NT5C, and SOX8. The importance of these genes was validated by quantitative real-time PCR. Collectively, our findings indicate that mitochondrial dysfunction may contribute to the damage that occurs to TM cells with a MYOCS341P background under mechanical stretch.© 2024 The Authors.