Glutamate excitotoxicity may contribute to the death of retinal ganglion cell (RGC) in glaucoma and other retinal diseases such as ischemia. Deubiquitinating enzyme (DUB) inhibitors are emerging as attractive targets for pharmacological intervention in neurodegenerative diseases. However, the role of PR-619, the broad spectrum DUB inhibitor, on RGCs under different stressful environment remains largely unknown. This study was designed to investigate the role of PR-619 in regulating mitophagy of RGCs under glutamate excitotoxicity. Primary cultured RGCs were incubated with PR-619 or vehicle control in the excitotoxicity model of 100 ┬ÁM glutamate treatment. Mitochondrial membrane potential was assessed by JC-1 assay. Cytotoxicity of RGCs was measured by LDH activity. Proteins levels of parkin, optineurin, LAMP1, Bax, Bcl-2 and the LC3-II/I ratio were analyzed by western blot. The distribution and morphology of mitochondria in RGCs was stained by MitoTracker and antibody against mitochondria membrane protein, and examined by confocal microscopy. We show here that in the presence of glutamate-induced excitotoxicity, PR-619 stabilized the mitochondrial membrane potential of RGCs, decreased cytotoxicity and apoptosis, attenuated the expression of Bax. Meanwhile, PR-619 promoted the protein levels of Bcl-2, parkin, optineurin, LAMP1 and the LC3-II/I ratio. While knockdown of parkin by siRNA diminished the neuroprotective effect of PR-619 on RGCs. These findings demonstrate that PR-619 exerted a neuroprotective effect and promoted parkin-mediated mitophagy on cultured RGCs against glutamate excitotoxicity. DUB inhibitors may be useful in protecting RGCs through modulating the parkin-mediated mitophagy pathway against excitotoxicity.