Supplementation of animal feed with iodine influences the iodine content of milk and therefore, in addition to salt iodination, provides another possibility for improving the human iodine supply. On the other hand, excessive iodine intake by humans through drinking milk must be avoided. Furthermore, the iodine content of milk varies, depending on the presence of iodine antagonists in feed (e.g., glucosinolates in rapeseed) and the applied iodine species. This study evaluated the impact of various feed iodine supplementation levels up to the permitted maximum level, the effect of applying rapeseed compared with a glucosinolate-free ration, and the impact of 2 different iodine species on the iodine content of milk. A total of 32 dairy cows were divided into 4 groups with 8 animals each. Two groups received distillers dried grains with solubles (DDGS) as the protein source, and the others received rapeseed meal (RSM, 16.5% of total diet). In each case, half the animals received feed supplemented with iodine in the form of potassium iodide, and the other half received feed supplemented with iodine in the form of calcium iodate. Iodine supplementation levels of 0, 0.5, 1, 2, 3, 4, and 5 mg/kg of dry matter (DM) were tested in consecutive periods of 21 d each. The milk iodine concentration increased with increasing iodine supplementation of the feed. Rapeseed meal in the ration (0.58 mmol of glucosinolates/kg of diet DM) diminished the milk iodine concentration by up to one-half to one-third of the concentration achieved by DDGS. At iodine supplementation levels of 2 mg/kg of DM and higher, the differences were significant. The application of iodate predominantly resulted in higher milk iodine concentrations compared with iodide, but not significantly in any period. At the highest tested iodine supplementation (5 mg/kg of DM), the milk iodine concentration increased up to 1,464 (iodide) and 1,578 microg/kg (iodate) when feeding DDGS and up to 718 (iodide) and 620 microg/kg (iodate) in the RSM groups. The carry over of iodine from feed into milk amounted to 30 to 56% when using DDGS, and 11 to 25% when using RSM. The maximum level of iodine currently allowed in the feed of dairy cows in Europe could lead to high milk iodine concentrations. As a result, the Tolerable Upper Intake Level in human nutrition could be exceeded. Therefore, this maximum level needs to be reevaluated. In addition to iodine supplementation, the application of RSM in the ration must be considered when estimating the iodine content of milk.