BackgroundDeinococcus radioduransis a robust bacterium that can withstand harsh environments that cause oxidative stress to macromolecules due to its cellular structure and physiological functions. Cells release extracellular vesicles for intercellular communication and the transfer of biological information; their payload reflects the status of the source cells. Yet, the biological role and mechanism ofDeinococcus radiodurans-derived extracellular vesicles remain unclear.AimThis study investigated the protective effects of membrane vesicles derived fromD. radiodurans(R1-MVs) against H2O2-induced oxidative stress in HaCaT cells.ResultsR1-MVs were identified as 322 nm spherical molecules. Pretreatment with R1-MVs inhibited H2O2-mediated apoptosis in HaCaT cells by suppressing the loss of mitochondrial membrane potential and reactive oxygen species (ROS) production. R1-MVs increased the superoxide dismutase (SOD) and catalase (CAT) activities, restored glutathione (GSH) homeostasis, and reduced malondialdehyde (MDA) production in H2O2-exposed HaCaT cells. Moreover, the protective effect of R1-MVs against H2O2-induced oxidative stress in HaCaT cells was dependent on the downregulation of mitogen-activated protein kinase (MAPK) phosphorylation and the upregulation of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. Furthermore, the weaker protective capabilities of R1-MVs derived from ΔDR2577 mutant than that of the wild-type R1-MVs confirmed our inferences and indicated that SlpA protein plays a crucial role in R1-MVs against H2O2-induced oxidative stress.ConclusionTaken together, R1-MVs exert significant protective effects against H2O2-induced oxidative stress in keratinocytes and have the potential to be applied in radiation-induced oxidative stress models.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12575-023-00211-4.