Optimal iodine intake during infancy is critical for brain development, but no estimated average requirement (EAR) is available for this age group. We measured daily iodine intake, excretion, and retention over a range of iodine intakes in early infancy to determine the minimum daily intake required to achieve iodine balance. In a dose-response crossover study, we randomly assigned healthy infants (n = 11; mean ± SD age 13 ± 3 wk) to sequentially consume over 33 d 3 infant formula milks (IFMs) containing 10.5, 19.3, and 38.5 μg I/100 kcal, respectively. Each IFM was consumed for 11 d, consisting of a 6-d run-in period followed by a 4-d balance period and 1 run-out day. Iodine intake (mean ± SD: 54.6 ± 8.1, 142.3 ± 23.1, and 268.4 ± 32.6 μg/d), excretion (55.9 ± 8.6, 121.9 ± 21.7, and 228.7 ± 39.3 μg/d), and retention (-1.6 ± 8.3, 20.6 ± 21.6, and 39.8 ± 34.3 μg/d) differed among the low, middle, and high iodine IFM groups (P < 0.001 for all). There was a linear relation between daily iodine intake and both daily iodine excretion and daily iodine retention. Zero balance (iodine intake = iodine excretion, iodine retention = 0 μg/d) was achieved at a daily iodine intake of 70 μg (95% CI: 60, 80 μg). Our data indicate the iodine requirement in 2- to 5-mo-old infants is 70 μg/d. Adding an allowance for accumulation of thyroidal iodine stores would produce an EAR of 72 μg and a recommended dietary allowance of 80 μg. This trial was registered at clinicaltrials.gov as NCT02045784.