To study the para- and transcellular permeability of columnar epithelium and follicle-associated epithelium of Peyer’s patches in the rat intestine, LPS was applied from the mucosal side to simulate the action of endotoxins from gram-negative bacteria of gut microbiota. LPS did not affect transepithelial resistance or sodium fluorescein permeability, but increased the levels of claudin-3 and claudin-4 in enterocytes, suggesting strengthening of the paracellular intestinal barrier. Transcellular permeability was evaluated by electron microscopy based on the number of vesicular structures in the cytoplasm of different cell types. LPS increased the number of small vesicles in follicle-associated epithelium of Peyers’ patches. In columnar epithelial cells, LPS reduced the number of smaller vesicles and increased the number of larger ones. LPS did not damage the tissue barrier, but enhanced transcytosis, which could potentiate the effects of endotoxin on its receptors in the intestinal mucosa. © Springer Science+Business Media, LLC, part of Springer Nature 2024.