Anatase TiO2 films are grown on Si (100) by atomic layer deposition. Three different interlayers (Si3N4, Al2O3, and Ti-rich SiOx) between the TiO2 films and the Si substrate have been considered. The band alignment of the titanium oxide films with the silicon substrate is investigated by x-ray photoelectron spectroscopy (XPS), internal photoemission (IPE) spectroscopy, and optical absorption (OA) measurements. XPS analysis indicates that TiO2/Si heterojunctions with different interlayers (ILs) have different valence-band offsets (VBOs). A VBO value of 2.56±0.09 eV is obtained for the TiO2/Ti-rich SiOx/Si sample. Similarly, we obtain a VBO value of 2.44±0.09 and 2.73±0.10 eV for the TiO2/Si3N4/Si and TiO2/Al2O3/Si samples, respectively. According to IPE and OA measurements, the band gap of the as-grown TiO2 films is 3.3±0.1 eV for all the samples. Combining the XPS and IPE data, the conduction band offset values at the TiO2/Si heterojunction are found to be -0.2±0.1, -0.4±0.1, and -0.5±0.1 eV for the TiO2/Si3N4/Si, TiO2/Ti-rich SiOx/Si, and TiO2/Al2O3/Si samples, respectively. According to our experimental results, the band alignment of a TiO2 film with the underlying Si (100) substrate is clearly affected by the presence of an IL, suggesting the possibility to tune the band structure of a TiO2/Si heterojunction by selecting the proper IL.