AbstractTraditional Transwell inserts with track‐etched 10 μm thick polymer membranes have been intensively used for studying cellular barriers. However, their thickness hampers direct cell‐cell interaction between the adjacent cells which has been shown to critically influence the barrier formation. Therefore, here the effect of reduced distance between the cells by using fivefold thinner (2 μm) optically transparent polydimethylsiloxane (PDMS) membranes is studied and compared with polycarbonate (PC) membranes. The authors validate their applicability as an alternative substrate for the study of the blood–brain barrier by performing a monoculture of brain endothelial cells (hCMEC/D3) and co‐culture with astrocytes in Transwells. The PDMS membranes supported the cellular protrusions through the well‐defined pores and allowed control over cellular transmigration by varying the pore size. Cellular localization of tight and adherens junction proteins ZO‐1, Claudin‐5, and VE‐cadherin is similar to PC membranes while their expression levels are affected as a function of membrane material and co‐culture with astrocytes. Additionally, a permeability assay indicated tighter barrier formation on the PDMS membrane. These results suggest the potential use of 2 μm PDMS membranes for in vitro modeling of biological barriers with improved co‐culture models and enhanced visibility of the cell culture.