Abstract This work examines the potential drug delivery barrier of the basement membrane (BM) by assessing the permeability of select macromolecules and nanoparticles. The study further extends to probing the effect of BM on intestinal epithelial cell attachment and monolayer characteristics, including cell morphology. Serum-free cultured Caco-2 cells were grown on BM-containing porous supports, which were obtained by prior culture of airway epithelial cells (Calu-3), shown to assemble and deposit a BM on the growth substrate, followed by decellularisation. Data overall show that the attachment capacity of Caco-2 cells, which is completely lost in serum-free culture, is fully restored when the cells are grown on BM-coated substrates, with cells forming intact monolayers with high electrical resistance and low permeability to macromolecules. Caco-2 cells cultured on BM-coated substrates displayed strikingly different morphological characteristics, suggestive of a higher level of differentiation and closer resemblance to the native intestinal epithelium. BM was found to notably hinder the diffusion of macromolecules and nanoparticles in a size dependent manner. This suggests that the specialised network of extracellular matrix proteins may have a significant impact on transmucosal delivery of certain therapeutics or drug delivery systems.