Various nanometer-sized multilayers were directly prepared onto the surface a mouse L929 fibroblast cells by a layer-by-layer (LbL) assembly technique to control the cell surface microenvironment and cell functions, such as viability, morphology, and proliferation. The species of LbL nanofilms strongly affected the cell morphology and growth. Polyelectrolyte (PE) multilayers induced a round-shaped morphology of the adhered cells, although each component of the multilayers had high cytocompatibility, whereas fibronectin (FN)-gelatin (G) and -dextran sulfate (DS) multilayers with EN-binding domain interactions (EN films) showed extended morphologies oldie cells similar to that of control cells (without films). A clear difference in cell proliferation was observed for PE and EN films. The cells with EN films on their surfaces showed good proliferation profiles independent of the film thickness, hut cell proliferation was not observed using the PE films although the cells survived during the culture period. Fluorescence microscopic and scanning electron microscopic observations clearly suggested a nanometer-sized meshwork morphology of the EN films on the cell surface after 24 h of incubation, whereas the PE films showed homogeneous film morphologies on the cell surface. These nanomeshwork morphologies seemed to be similar to the fibrous structure of natural extracellular matrix. The results of this study demonstrated that the components, charge, and morphology of LbL nanofilms prepared directly on the cell surface strongly affected cell functions, and the effects of these LbL nanofilms on cell functions differed vastly as compared to PE films prepared on a substrate. The preparation of LbL nanofilms onto a cell surface might be a novel and interesting technique to control cell functions.