Iodine is an essential element for humans; however, it can be toxic depending on its chemical form. A variety of toxic and non-toxic iodine species have been identified in environmental water and in the drinking water produced by public water treatment plants. Here, we examined the change of iodine species during the water treatment process at a public water treatment plant in Japan. Samples of raw water and of treated water immediately after each of eight treatment stages comprising the treatment process were collected, and a speciation analysis was conducted by means of ion chromatography– and size-exclusion chromatography–inductively coupled plasma mass spectrometry. In the raw water, iodine was found mainly as iodide or iodinated humic substances that were ultimately oxidized and transformed into iodate, a form non-toxic to humans, by two independent oxidation stages in the water treatment process – ozone treatment and chlorine treatment. No disinfection byproducts were detected at any stage of the treatment process. Fluorescence spectrometry with multivariate analysis revealed that humic substances were markedly decreased by ozone treatment, but not by chlorine treatment. The present results show that, at the plant-scale, ozone treatment is an effective means of removing toxic iodine species from raw water.