At recent years, the impairment caused by iodine excess are paid more attention. However, there is still largely unknown about the exact mechanism induced by excessive iodine. MiRNAs have been found to act as biomarkers for a variety of diseases, whereas fewer studies focused on miRNAs related to a cluster of genes regulating thyroid hormone synthesis, such as NIS, Pendrin, TPO, MCT8, TSHR, TSH alpha, and TSH beta-related miRNAs in structural and functional changes of the thyroid gland induced by subchronic and chronic high iodine exposure. In the present study, one hundred and twenty 4-week-old female Wistar rats were randomly divided into control group (I-50 mu g/L KIO3); HI 1 (I-6000 mu g/L KIO3); HI 2 (I-10000 mu g/L KIO3); and HI 3 (I-50000 mu g/L KIO3), the exposure period was 3 months and 6 months, respectively. The iodine contents in the urine and blood, thyroid function, and pathological changes were determined. In addition, levels of thyroid hormone synthesis genes and the associated miRNAs profiling were detected. The results showed that subclinical hypothyroidism occurred in the high iodine groups with subchronic high iodine exposure, while 6-month exposure led to hypothyroidism in the I-10000 mu g/L and I-50000 mu g/L groups. Subchronic and chronic high iodine exposure caused mRNA and protein levels of NIS, TPO, and TSHR decreased significantly, and Pendrin expression increased significantly. In addition, MCT8 mRNA and protein levels are only remarkably decreased under the subchronic exposure. PCR results showed that levels of miR-200b-3p, miR-185-5p, miR-24-3p, miR-200a-3p, and miR-25-3p increased significantly exposed to high iodine for 3 months, while miR-675-5p, miR-883-5p, and miR-300-3p levels increased significantly under the exposure to high iodine for 6 months. In addition, miR-1839-3p level was markedly decreased exposed to high iodine for 3 and 6 months. Taken together, the miRNA profiling of genes regulating thyroid hormone synthesis remarkably altered from subclinical hypothyroidism to hypothyroidism induced by excess iodine exposure, and some miRNAs may play an important role in subclinical hypothyroidism or hypothyroidism through regulating NIS, Pendrin, TPO, MCT8, and TSHR providing promising targets to alleviate the impairment on the structure and function of thyroid gland.