Bacterial extracellular vesicles (EVs) have emerged as a relevant mode of communication between host and resident microbiota. Evidence is limited regarding the ability of Gram-positive EVs to interact with primary colonic epithelial cells. We propose a murine organoid-based in vitro model to study the impact of EVs from the lactic acid bacteria species Limosilactobacillus reuteri (LR) and Lactiplantibacillus plantarum (LP) on primary colonic epithelium. Previous EV characterization revealed significant differences in zeta-potential (LR: −0.0185 V vs. LP: −0.0103 V, p 0.0125) and particle concentration (LR: 3.38 × 10⁸ particles/mL vs. LP: 8.32 × 10⁸ particles/mL, p 0.0029) but not in lipoteichoic acid (LTA) content and long-chain fatty acid composition. Applied to colonic organoids, uptake efficiency differed between LR and LP EVs, with LR exhibiting higher internalization rates than LP (5 µg EV, 46.56% vs. 20.03%, p < 0.0001) at the expense of cellular viability. Internalized EVs partially co-localized with endo-lysosomal compartments and, depending on the bacterial species of origin, induced a proteomic stress response mimicking molecular features of neurodegenerative diseases. Proteomic profiling of EVs and parental bacteria revealed the existence of lipoproteins predicted to be embedded in the cytoplasmic membrane, potentially acting as adhesion factors to the epithelium. Pre-emptive blocking with LTA reduced EV uptake into the epithelium (1 µg/mL LTA: 3.77 × 10⁵ A.U. vs. control: 1.02 × 10⁶ A.U., p 0.0231). Efficiency of EV internalization differs between LR and LP and may be associated with LTA decoration. Studies using LTA-mutant Lactobacillus species are warranted to clarify the role of vesicle-bound LTA in the EV uptake machinery.