To evaluate the effect of electrical conductivity of biomaterials on osteogenesis, polypyrrole (PPy) was fabricated by oxidative chemical polymerization as substrates for cell culture. Through adjusting the concentrations of monomer and initiator, polypyrrole films with different electrical conductivities were fabricated. These fabricated polypyrrole films are transparent enough for easy optical microscopy. Fourier transform infrared spectroscopy, X-ray spectroscopy and four-point probe were used to assess the microstructures, surface chemical compositions and electrical sheet resistance of films, respectively. Results indicate that higher monomer and initiator concentration leads to highly-branched PPy chains and thus promotes the electron mobility and electrical conductivity. Selected polypyrrole films then were applied for culturing rat bone marrow stromal cells. Cell viability and mineralization assays reveal that not only these films are biocompatible, but also capable of enhancing the calcium deposition into the extra cellular matrix by the differentiated cells.