The formation of crystalline titanium dioxide (TiO2) nanoparticles in Nafion membrane structure was investigated. The X-ray powder diffraction and high-resolution transmission electron microscopy (TEM) results show that these nanoparticles are pure anatase TiO2. The TEM results also show that the crystalline TiO2 nanoparticles are ellipse in shape, with an estimated average aspect ratio (long axis/short axis) of 1.4. The average size of the TiO2 nanoparticles, which is independent of the loading of titanium, is similar to the estimated dimensions of the hydrophilic cavities in Nafion membrane in terms of the ion cluster model. However, there is no evidence for any TiO2 nanoparticles in the proposed channels that connect the hydrophilic cavities. The fact that the TiO2 nanoparticles are distributed homogeneously throughout the Nafion membrane structure suggests that the nanoparticles-embedded membrane still maintains a certain degree of porosity. The properties and potential applications of the Nafion-supported crystalline TiO2 nanoparticles are discussed.