Various electrochemical measurements were performed on titanium with and without culturing murine fibroblasts L929 to characterize the effects of cells on interface electrochemical properties between titanium and cells. Open-circuit potential of titanium decreased with L929 cells, which was caused by the shift of equilibrium potential between cathodic and anodic reactions indicated by the decrease in cathodic current density with L929 cells. In cathodic potential step test, the decrease in current density following to the peak current density was delayed with L929 cells, indicating that diffusivity of molecules and ions decreased with the cells. In addition, alternating current impedance measurement and data approximation to the electrical equivalent circuit model revealed that the circuit element for diffusion resistance of biomolecule adsorption layer increased with L929 cells. Consequently, the effect of cells on the interface property is the retardation of diffusion through the biomolecule adsorption layer due to the increase in biomolecule density with extracellular matrix consisting with proteins and glycosaminoglycans generated by the cells.