Purpose: Initial adhesion of cells to implant surfaces and subsequent behavior of the cells are important determinants for biocompatibility of the implants. It was previously reported that both adhesion of MC3T3-E1 osteoblast-like cells to titanium (Ti) plates and their differentiation into more mature cells on the plates were stimulated by treatment of the plates with glow discharge plasma (GDP). However, the mechanisms of these processes have not yet been identified. In this study, the adhesion and differentiation mechanism of osteoblast-like cells to Ti with and without GDP were investigated. Materials and Methods: The adhesion and differentiation mechanism of MC3T3-E1 osteoblast-like cells to Ti, with and without GDP, were investigated by cultivation in serum-free medium and use of a competitive inhibition test to examine the influence of extracellular matrix proteins contained in the serum and to identify cell binding proteins. In addition, the amount of fibronectin adsorption on each Ti plate was investigated by enzyme-linked immunosorbent assay and fluorescein isothiocyanate labeling. Furthermore, the stress fiber formation and morphology of cells on each plate were evaluated microscopically. Results: Adherent cells on Ti plates, with and without GDP, were significantly reduced in serum-free conditions and the presence of RGDS (Arg-Gly-Asp-Ser) peptides. Fibronectin adsorption on titanium plates was increased by GDP. Furthermore, stress fiber formation of cells was extremely progressive on the Ti plates treated with GDP and was not observed on the cells inhibited by RGDS peptide. Discussion: These results suggest that RGDS containing serum proteins have a major role in regulating specific adhesion of cells to Ti, and GDP promoted cell adhesion and differentiation on Ti by increasing the adsorption of proteins. Conclusion: According to this study, the adhesion and differentiation mechanism of osteoblast-like cells to Ti, with and without GDP, can be obtained.