Objective To investigate the dynamic changes and significance of thyroid hormones [triiodothyronine (T3), thyroxine (T4), reverse triiodothyronine (rT3)] in the liver, kidney, and hippocampus of iodine-deficient rats. Methods Thirty-six SD rats were randomly assigned to three intervention time points (4, 8, and 16 weeks). At each time point, two groups (n=6 per group) were established: the adequate iodine (NI) group and the iodine-deficient (ID) group. All rats were fed an iodine-deficient diet (iodine content <50 μg/kg), and iodine intervention was achieved by adjusting the concentration of potassium iodide (KI) in the drinking water. At the end of the intervention, rat liver, kidney, and hippocampal tissues were collected using cryovials. Serum was collected from abdominal aorta blood. Thyroid function was assessed by chemiluminescence immunoassay. Thyroid hormone levels in the liver, kidney, and hippocampus tissues were measured by ELISA, the expression of deiodinase (Dio) expression was analyzed by Western blot. Results Compared with the NI group, the ID group exhibited a compensatory increase in serum thyroid stimulating hormone (TSH) during the middle and late stages (P<0.01), along with a persistent decrease in free thyroxine (FT4) after week 4 feeding (P<0.05). Free triiodothyronine (FT3) levels showed a significant increase at week 8 (P<0.01) followed by a significant decrease at week 16 (P<0.01). Compared with the NI group, the ID group exhibited significantly decreased T3 levels in the liver during the early stage and decreased T4 levels during the late stage. Hepatic Dio1 expression was significantly upregulated after week 8 feeding, while Dio3 expression was continuously inhibited during the early and middle stages (all P < 0.01). In the kidney, T3, T4, and rT3 levels were significantly reduced at week 16 in the ID group (P<0.01). Renal Dio1 expression was elevated at week 8, whereas Dio3 expression was persistently inhibited after week 8 feeding (all P < 0.01). In the hippocampus, T3 and T4 levels were significantly decreased at multiple time points in the ID group (P<0.05). Hippocampal Dio2 expression initially decreased in the early stage and then increased by week 8, while Dio3 expression increased from the early stage to the late stage (all P<0.01). Conclusion Under chronic iodine deficiency, the liver and kidney response by modulating Dio1 and Dio3 expression to promote peripheral T3 secretion for systemic metabolic needs. Conversely, the hippocampus maintains cerebral T3 stability via bidirectional regulation of Dio2 and Dio3 expression, thereby preserving normal neural function.