Photocatalytic reaction of methanol on an anatase TiO2(001)-(1 × 4) reconstructed surface, a prototype reaction for photocatalysis, was studied by means of X-ray photoelectron spectroscopy, thermal desorption spectrum, and density functional theory calculations. Photocatalytic oxidation reaction was observed to exclusively occur at the Ti4Csites of the (1 × 4) added row but not at the Ti5Csites of the (1 × 1) basal surface. The accompanying density functional theory calculation results demonstrate that the valence band maximum is localized at the oxygen atoms of the (1 × 4) added row and the methoxy species bonded to the Ti4Csites, respectively, for the clean and methanol-covered anatase TiO2(001)-(1 × 4) surfaces. This leads to a Ti4Csite-specific oxidation of the methoxy species by photogenerated holes. These results reveal a concept of surface reconstruction-induced site-specific charge separation and photocatalytic reaction on oxide photocatalysts that will greatly deepen the understanding of the vital role of oxide surface structure in photocatalytic reactions.