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The extensive use of antibiotics has been a worldwide environmental issue. In this study the fate of oxytetracycline (OTC), under photoirradiation, is investigated. The results show that OTC photolysis follows first order model kinetics. Direct photolysis rate is found to be dependent on the initial OTC concentration, with a k value ranging from 0.0075 to 0.0141 min-1, in the OTC concentration from 40 to 10 mg/L. OTC photolysis is highly pH-dependent and strongly enhanced at a high pH value, with a k value of 0.0629 min-1 at pH 9. Enhanced OTC photolysis has also been observed in the presence of nitrate and low concentration of dissolved organic matter. Upon irradiation for 240 min, only 13.5% reduction of TOC occurs in spite of a rapid consumption of 90% of OTC. The byproducts from OTC photolysis have been analyzed using high-performance liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), and the degradation pathway of OTC in the photo process is proposed. By employing luminescent bacterium to assess the adversity of the degradation compounds, an increased effect of toxicity occurs in spite of the great consumption of OTC in the photoirradiation process. After irradiation for 240 min, the inhibition rate is 47%, significantly higher than the initial rate of 21% (p < 0.05), revealing a potentially higher adversity risk on the microorganism upon OTC photolysis.