Controlling the cell behavior on biocompatible polymer surfaces is critical for the development of suitable medical implant coatings as well as in anti-adhesive applications. Synthetic glycopolymer brushes, based on sugar methacrylate monomers have been reported as robust surfaces to resist protein adsorption and cell adhesion. In this study, poly(D-gluconamidoethyl methacrylate) (PGAMA) brushes of various chain lengths were synthesized directly from initiator functionalized glass substrates using surface-initiated atom transfer radical polymerization. The glycopolymer film thicknesses were determined by ellipsometry, whereas the wettability and the morphology of the surfaces were characterized by static water contact angle measurements and atomic force microscopy, respectively. Stable, grafted films with thicknesses in the dry state between 4 and 20 nm and of low roughness (~1 nm) were obtained by varying the polymerization time. Cell experiments with MC3T3-E1 pre-osteoblasts cultured on the PGAMA brushes were performed to examine the effect of film thickness on the cell morphology, cytoskeleton organization and growth. The results revealed good cell spreading and proliferation on PGAMA layers of low film thickness, whereas cell adhesion was prevented on polymer films with thickness higher than ~10 nm, indicating their potential use in medical implants and anti-adhesive surfaces, respectively.