Stimulation with recombinant human thyroid-stimulating hormone (rhTSH) before radioactive iodine administration for patients with thyroid cancer may increase the body iodine pool in the presence of continued levothyroxine; however, the precise significance of its influence remains unclear.This was a prospective observational study conducted between March 2017 and August 2020. We measured the 24-hour urinary iodine excretion and urinary iodine-to-creatinine ratio in patients with thyroid cancer stimulated by rhTSH or thyroid hormone withdrawal (THW) before radioactive iodine administration. Oral iodine intake was controlled by a 7-day self-managed low iodine diet, followed by a strict 3-day low iodine diet while in the hospital.Overall, 343 subjects were included (rhTSH: n = 181; THW: n = 162). The mean levothyroxine dose in the rhTSH group was 115.2 μg daily. The median 24-hour urinary iodine and urinary iodine-to-creatinine ratio in the rhTSH group (71.0 [interquartile range, 57.5-88.0] μg/day and 80.0 [59.0-97.5] μg/gCr, respectively) were significantly higher than those in the THW group (42.0 [30.0-59.0] μg/day and 39.0 [28.0-61.3] μg/gCr, respectively; both P.001). After propensity score matching by age, sex, body weight, and renal function (rhTSH: n = 106; THW: n = 106), consistent results for both values were observed for both methods. The increase in urinary iodine with the rhTSH method was smaller than the expected value calculated from the amount of levothyroxine.Urinary iodine excretion was significantly higher among patients with rhTSH stimulation than those with THW, indicating that the rhTSH method slightly increases the body iodine pool.