Inflammatory bowel disease (IBD) is a chronic intestinal disorder characterized by barrier dysfunction, for which effective treatments are still lacking. Plant-derived extracellular vesicles represent an emerging class of natural nanotherapeutics. In this study, we isolated and characterized extracellular vesicles from barley leaf (BLEVs), demonstrating typical vesicular morphology with an average diameter of 177 nm and negative surface charge. BLEVs exhibited excellent biosafety, stability, and colon-targeting capability upon oral administration. In mouse models of colitis induced by dextran sulfate sodium or Citrobacter rodentium, BLEVs significantly restored intestinal barrier integrity. Using colonic organoids, we further showed that BLEVs promote intestinal stem cell proliferation and epithelial regeneration through activation of the retinoic acid (RA) metabolism pathway. Critically, pharmacological inhibition of retinaldehyde dehydrogenase (RALDH) abolished the protective effects of BLEVs, identifying RALDH as a key molecular target. These findings position BLEVs as a promising therapeutic strategy for IBD, highlighting RA signaling as a potential target for epithelial homeostasis restoration.