Electrospining method was used to prepare bioactive TiO2 fibers films in this study. The acetic acid/ethanol/tetrabutyl titanate/polyvinylpyrrolidone (PVP) solvent system was used as precursor for the electrospining. The TiO2 fiber structures (including its fiber diameter, morphology, and phase composition) could be controlled by changing feeding rate, PVP concentration and sinter temperature. The fiber films were subjected to simulated body fluid soaking experiments and MG63 cells culture experiments to study their bioactivity. According to the X-ray diffraction and MTT assay results, the fiber containing with anatase showed better apatite formation ability than that without anatase at the early stage, while cell proliferated on anatase-rutile TiO2 fiber was better than that on other samples (p < 0.05).Some string beads in the fiber were beneficial for apatite formation, while the cell proliferated best on the fiber film without string beads (p < 0.05). The fiber with a diameter of 200 nm had the best apatite formation ability and osteoblast compatibility (p < 0.05). The results showed that the TiO2 fiber film structure had great influence on its bioactivity. It indicated that the electronspining method is an effective way to prepare bioactive titania fiber films, and it is possible to control the structure of the films in the spinning process to optimize the bioactivity of TiO2 fiber. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A:64–74, 2013.