In tissue engineering, surface characteristics of a biomaterial are one of most important factors determining the compatibility with the environment. They influence attachment and growth of cells onto the material. In many cases, the surface should to be modified and engineered in the desired direction. The modification of non-adhesive surfaces with polyelectrolyte multilayer films (PMF) was recently depicted as a powerful technique to promote the growth of different cell lines. In this study, we evaluated the possible use of two different PMF as surface modification for the culture of mesenchymal stem cells (MSC). We used two types of PMF which differed by the nature of the initial anchoring layer which was poly(ethylenimine) (PEI) or poly(allylamine hydrochloride) (PAH). This initial polyelectrolytes adsorption was followed by the alternated deposition of poly(sodium 4-styrenesulfonate) (PSS) and (PAH) in order to obtain a PEI-(PSS-PAH)(3) film or a PAH-(PSS-PAH)(3) film. In order to control the behaviour of MSC, the cell viability was evaluated by Alamar Blue assay and the actin cytoskeleton was labelled and visualised in a confocal microscope. The behaviour of cells on the two PMF was compared to cells cultivated on surfaces treated with fibronectin. The results showed that PAH-(PSS-PAH)(3) PMF improve the growth of cells, inducing a higher cell viability compared to PEI-(PSS-PAH)(3) PMF and fibronectin at 2, 3 and 7 days of culture. Moreover, those cells showed a well-organized actin cytoskeleton. In conclusion, PAH-(PSS-PAH)(3) polyelectrolyte multilayer film seems to constitute an excellent material for MSC seeding.