Azithromycin (AZI) has been listed as an emerging contaminant by the US EPA since 2009 because it is frequently detected in wastewater, surface water, and drinking water. In this paper, the chlorination of AZI in drinking water was simulated and studied. The results indicated that new compounds were generated in the chlorination of AZI. The byproducts were identified by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF), and four of the byproducts were detected in real water samples. The kinetic studies demonstrated that the reaction rates of AZI chlorination were dependent on the initial concentration of free chlorine and the pH value. The potential toxicities of the byproducts were assessed by quantitative structure activity relationship software and investigated by the viability of Chinese hamster lung (CHL), Jurkat T and Hep G2 cells.