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Surface engineering is a critical effort in defining substrates for cell culture and tissue engineering. In this context, multilayer self-assembly is an attractive method for creating novel composites with specialized chemical and physical properties that is currently drawing attention for potential application in this area. In this work, effects of thickness, surface roughness, and surface material of multilayer polymer nanofilms on the growth of rat aortic smooth muscle cells were studied. Polyelectrolyte multilayers (PEMs) electrostatically constructed from poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) (PSS) with gelatin, fibronectin, and PSS surface coatings were evaluated for interactions with smooth muscle cells (SMCs) in an in vitro environment. The results prove that PEMs terminated with cell-adhesive proteins promote the attachment and further growth of SMCs, and that this property is dependent upon the number of layers in the underlying multilayer film architecture. Cell roundness and number of pseudopodia were also influenced by the number of layers in the nanofilms. These findings are significant in that they demonstrate that both surface coatings and underlying architecture of nanofilms affect the morphology and growth of SMCs, which means additional degrees of freedom are available for design of biomaterials. This work supports the excellent potential of nanoassembled ultrathin films for biosurface engineering, and points to a novel perspective for controlling cell-material interaction that can lead to an elegant system for defining the extracellular in vitro environment.