Microglial activation plays a pivotal role in the pathogenesis of retinal ganglion cell (RGC) degeneration resulting from optic nerve crush (ONC). Small extracellular vesicles (sEVs) secreted by mesenchymal stem cells (MSCs) have the potential to prevent retinal degeneration by modulating microglial activation. In this study, we elucidated the specific effects of sEVs derived from IFN-γ-primed MSCs on the phenotypic transition of microglia and the associated pathways in ONC mice. The ONC mice model was established and administered intravitreal injection with the sEVs derived from native MSCs (native sEVs) and the sEVs derived from MSCs primed with IFN-γ (IFNγ-sEVs). Their respective effects on the survival of the retinal ganglion cells (RGCs) and the transition of microglia phenotypes were determined through visual function testing and immunohistochemical staining. Combined with mRNA seq and microRNA seq techniques, we elucidated the mechanism of modulation of microglia phenotypic transformation by sEVs derived from MSCs primed by IFNγ. It demonstrated that IFNγ-sEVs exhibited superior protective effects against RGC loss and reduced inflammatory responses in the ONC retina compared to native sEVs. Both types of sEVs promoted microglia activation to disease-associated microglia (DAM) phenotype, while IFNγ-sEVs especially suppressed interferon-responsive microglia (IRM) activation during RGCs degeneration. Subsequent miRNA sequencing suggested that , which exhibited the most significant differential expression between the two sEVs types and elevated expression in IFNγ-sEVs, inhibited the expression of IRM and ribosomal genes. These findings suggest that IFN-γ-preconditioned MSCs may enhance sEVs of neuroprotection on RGCs by suppressing IRM activation through the secretion of sEVs containing specific microRNAs in ONC mice.