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Wild ginseng is known to have better pharmacological effects than cultivated ginseng. Additionally, recently developed bioengineering technology has made it possible to produce cultured wild ginseng with the same genetic composition. In this study, we investigated the change in characteristics and the improvement of the intestinal barrier of cultured wild ginseng roots (CWG) and fermented cultured wild ginseng roots (FCWG). First, we screened nine strains of bacteria that are capable of growing on 5-brix CWG medium, and Limosilactobacillus fermentum HY7303 (HY7303) showed the highest growth. Second, changes in the characteristics of CWG due to fermentation using HY7303 showed that pH and total carbohydrates decreased, and reducing sugars increased. The contents of minor ginsenosides (Rg3(s), Rk1, and Rg5) increased. Third, extracellular vesicles (EVs) with a single peak at 493.7 nm were isolated from CWG, and EVs with three peaks at 9.0 nm, 155.6 nm, and 459.0 nm were isolated from FCWG, respectively. Finally, when we treated Caco-2 cells with FCWG and EVs, we confirmed the improvement of intestinal barrier functions, including recovery, permeability, and expression of tight-junction protein genes. In this study, we confirmed the potential pharmacological effects of minor ginsenosides and EVs derived from FCWG. In conclusion, this study suggests that CWG fermentation with HY7303 improves the intestinal barrier by increasing minor ginsenosides and producing EVs. [ABSTRACT FROM AUTHOR]