Mitochondrial metabolism in the trabecular meshwork (TM) plays a critical role in maintaining intraocular pressure homeostasis by supporting the energy-demanding processes involved in aqueous humour outflow. In primary open-angle glaucoma, oxidative stress impairs mitochondrial function, leading to TM dysfunction. Therefore, understanding and targeting mitochondrial health in TM cells could offer a novel therapeutic strategy. Pyrroloquinoline quinone (PQQ) is a redox cofactor with antioxidant and mitochondrial-enhancing properties. However, its effects on human TM (HTM) cells remain largely unexplored. This study examined PQQ cytoprotective effects against H₂O₂-induced oxidative stress in HTM cells. Seahorse analyses revealed that PQQ alone improves mitochondrial respiration and ATP production. Moreover, PQQ mitigates H₂O₂-induced cellular damage and preserves mitochondrial function by normalising proton leak and increasing ATP levels. Furthermore, TEM and confocal microscopy showed that PQQ can partially alleviate structural damage, restoring mitochondrial network morphology, thereby leading to reduced cell death. Although these protective effects seem not to be mediated by changes in mitochondrial content or activation of the SIRT1/PGC1-α pathway, they may involve modulation of SIRT3, a key factor of mitochondrial metabolism and homeostasis. Overall, these results suggest that PQQ may represent a promising candidate for restoring mitochondrial function and reversing oxidative damage in HTM cells.