There is a research gap on the transformation of ofloxacin (OFL) in water supply systems under the action of ClO2. The degradation kinetics of OFL under different ClO2 concentrations, pH and pipe materials, and formation of intermediates, as well as the toxicity of water in water supply systems were first studied. The results showed that the degradation of OFL in PE pipe and deionized water increased with the increase of ClO2 concentration, and the reaction of OFL followed the second-order kinetic model. The removal rate of OFL in deionized water was faster than that in PE pipe. The degradation rate of OFL in PE tube and deionized water were positively correlated with pH. In addition, degradation efficiency of OFL in different pipes followed: cast iron pipe > stainless steel pipe > PE pipe. The detection of OFL intermediates showed that the cleavage of piperazine groups were the main and initial products in the degradation process of OFL. In addition, the decarboxylation intermediates of quinolone groups were also detected. The aquatic toxicity test showed that the degradation of OFL by ClO2 would increase the toxicity of water, therefore, the decrease of OFL may not mean the decrease of water toxicity.