Intensified research interests are posed with the thionudeobase 4-thiouracil (4-TU), due to its important biological function as site-specific photoprobe to detect RNA structures and nucleic acid-nucleic acid contacts. By means of time-resolved IR spectroscopy and density functional theory (DFT) studies, we have examined the unique photophysical and photochemical properties of 4-TU. It is shown that 4-TU absorbs UVA light and results in the triplet formation with a high quantum yield (0.9). Under N-2-saturated anaerobic conditions, the reactive triplet undergoes mainly cross-linking, leading to the (5-4)/(6-4) pyrimidine-pyrimidone product. In the presence of O-2 under aerobic conditions, the triplet 4-TU acts as an energy donor to produce singlet oxygen O-1(2) by triplet-triplet energy transfer. The highly reactive oxygen species O-1(2) then reacts readily with 4-TU, leading to the products of uracil (U) with a yield of 0.2 and uracil-6-sulfonate (U-SO3) that is fluorescent at similar to 390 nm. The product formation pathways and product distribution are well rationalized by the joint B3LYP/6-311+G(d,p) calculations. From dynamics and mechanistic point of views, these results enable a further understanding for 4-TU acting as reactive precursors for photochemical reactions relevant to O-1(2), which has profound implications for photo cross-linking, DNA photodamage, as well as photodynamic therapy studies.