Background: Acetaminophen (APAP) overdose is the common cause of acute liver failure (ALF) due to the oxidative damage of multiple cellular components. This study aimed to investigate whether plasma membrane vesicles (PMVs) from human umbilical cord mesenchymal stem cells (hUCMSCs) could be exploited as a novel stem cell therapy for APAP-induced liver injury. Methods: PMVs from hUCMSCs were prepared with an improved procedure including a chemical enucleation step followed by a mechanical extrusion. PMVs of hUCMSCs were characterized and supplemented to hepatocyte cultures. Rescue of APAP-induced hepatocyte damage was evaluated. Results: The hUCMSCs displayed typical fibroblastic morphology and multipotency when cultivated under adipogenic, osteogenic, or chondrogenic conditions. PMVs of hUCMSCs maintained the stem cell phenotype, including the presence of CD13, CD29, CD44, CD73, and HLA-ABC, but the absence of CD45, CD117, CD31, CD34, and HLA-DR on the plasma membrane surface. RT-PCR and transcriptomic analyses showed that PMVs were similar to hUCMSCs in terms of mRNA profile, including the expression of stemness genes GATA4/5/6, Nanog, and Oct1/2/ 4. GO term analysis showed that the most prominent reduced transcripts in PMVs belong to integral membrane components, extracellular vesicular exosome, and extracellular matrix. Immunofluorescence labeling/staining and confocal microscopy assays showed that PMVs enclosed cellular organelles, including mitochondria, lysosomes, proteasomes, and endoplasmic reticula. Incorporation of the fusogenic VSV-G viral membrane glycoprotein stimulated the endosomal release of PMV contents into the cytoplasm. Further, the addition of PMVs and a mitochondrial-targeted antioxidant Mito-Tempo into cultures of APAP-treated HepG2 cells resulted in reduced cell death, enhanced viability, and increased mitochondrial membrane potential. Lastly, this study demonstrated that