Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin, classified as the type III intermediate filament family, maintain the integrity and architecture of various cell types. Recently, we reported their cell surface expression and binding to multivalent N‐acetylglucosamine‐conjugated polymers. Furthermore, the presence of vimentin on the surface of various cell types including malignant tumor cells and fibroblasts has been demonstrated. Type III intermediate filament proteins are traditionally considered intracellular proteins and do not possess signal peptides for cell membrane recruitment. Therefore, the mechanism of their transport to the cell surface is unclear. In the current study, we aimed to elucidate this mechanism by focusing on the relationship between their multimeric structure and lipid bilayer affinity. Blue native polyacrylamide gel electrophoresis demonstrated that cell surface‐expressed type III intermediate filament proteins formed a multimeric mostly including 4–12‐mers but not filamentous structure. Moreover, surface plasmon resonance analysis revealed that the multimeric structure of these recombinant proteins had high affinity to lipid bilayers, whereas their filament‐like large multimeric structure did not. Our results suggest that type III intermediate filaments are incorporated into the cell membrane through alteration from a filamentous to a multimeric structure.