This study employed the medium pressure UV/peroxymonosulfate (MPUV/PMS), a new sulfate radical-based advanced oxidation process, to eliminate tetracycline (TTC) in water. At pH = 3.7, initial TTC concentration of 11.25 μM, PMS dosage of 0.2 mM and UV dose of 250 mJ cm−2, 82% of TTC was degraded by MPUV/PMS. The second-order reaction rate constants of TTC with SO4− and OH were found to be 1.4 × 1010 M−1 s−1 and 6.0 × 109 M−1 s−1, respectively. Radical quenching experiments indicated that OH played the major role in the degradation of TTC. Higher PMS dosage (0.1–1.0 mM) and higher pH (3–11) could accelerate the TTC removal. Besides, the presence of Cl− (0.1–5.0 mM) and CO32− (0.05–0.5 mM) could also promote the reaction. Eight transformation products (TPs) were identified, and the potential degradation pathways mainly involved hydroxylation, demethylation and decarbonylation processes. The variation in the genotoxicity was investigated using the umu-test, and the results indicate that the genotoxicity of TTC after the MPUV/PMS treatment significantly increased during the initial stage. In addition, the ecotoxicity and mutagenicity of TTC and its TPs were predicted using quantitative structure-activity relationship (QSAR) analysis, and the results revealed that some TPs could have equivalent and even higher toxicity than TTC. MPUV/PMS showed better performance in TTC degradation in real waters than in Milli-Q water. MPUV/PMS is concluded to be an efficient method for removing TTC, but more attention should be paid to the changes of toxicity during this process.