As an end metabolism product of the widely used thiopurine drugs, 6-thioguanine (6-TG) absorbs UVA and produces (1)O2 by photosensitization. This unusual photochemical property triggers a variety of DNA damage, among which the oxidation of 6-TG itself by (1)O2 to the promutagenic product guanine-6-sulfonate (G(SO3)) represents one of the major forms. It has been suspected that there exists an initial intermediate, G(SO), prior to its further oxidation to G(SO2) and G(SO3), but G(SO) has never been observed. Using density functional theory, we have explored the energetics and intermediates of 6-TG and (1)O2. A new mechanism via G(SOOH) → G(SO2) → G(SO4) → G(SO3) has been discovered to be the most feasible energetically, whereas the anticipated G(SO) mechanism is found to encounter an inaccessibly high barrier and thus is prevented. The mechanism through the G(SOOH) and G(SO4) intermediates can be validated further by joint experimental measurements, where the fast rate constant of 4.9 × 10(9) M(-1) s(-1) and the reaction stoichiometry of 0.58 supports this low-barrier new mechanism. In addition to the dominant pathway of G(SOOH) → G(SO2) → G(SO4) → G(SO3), a side pathway with higher barrier, G(SOOH) → G, has also been located, providing a rationalization for the observed product distributions of G(SO2) and G(SO3) as major products and G as minor product. From mechanistic and kinetics points of view, the present findings provide new chemical insights to understand the high phototoxicity of 6-TG in DNA and point to methods of using 6-TG as a sensitive fluorescence probe for the quantitative detection of (1)O2, which holds particular promise for detecting (1)O2 in DNA-related biological surroundings.