Purpose Thyroid concentrates radioactive iodine by sodium-iodide symporter; this is used for treating hyperthyroidism and thyroid cancer. Pancreas expresses NIS and radioactive iodine uptake may damage pancreatic beta-cells and predispose patients to type 2 diabetes. The aim of this study was to determine whether radioactive iodine is associated with glucose metabolism in thyroidectomized rats. Methods Forty male Wistar rats were divided into four groups (n = 10/each); control, thyroidectomized, thyroidectomized-treated with 131-I (TX+I), and thyroidectomized-treated with 131-I and l-thyroxine (TX+I+T-4). At the end of study, serum fasting glucose, insulin, thyroid-stimulating hormone, and free tetraiodothyronine were measured, intraperitoneal glucose tolerance test was performed, and homeostasis model assessment-insulin resistance was calculated. In in vitro experiments, glucose-stimulated insulin secretion from pancreatic islets and sodium-iodide symporter mRNA expression in thyroid and islets were determined. Results Compared to control group, free tetraiodothyronine was lower by 41 and 77% and thyroid-stimulating hormone was higher by 36 and 126% in thyroidectomized and TX+I groups, respectively. Compared to controls, rats in TX+I group had glucose intolerance as assessed using the area under curve of intraperitoneal glucose tolerance test (12,376 +/- 542 vs. 20,769 +/- 1070, P < 0.001) and l-thyroxine replacement therapy restored the value (14,286 +/- 328.24) to near normal. Fasting insulin and homeostasis model assessment-insulin resistance were comparable in all groups, however fasting glucose was higher in TX+I group. In in vitro experiments, glucose-stimulated insulin secretion from islets did not differ between groups. Conclusion Radioactive iodine therapy per se had no effect on glucose metabolism, just intensified thyroid hormone deficiency and the alterations on glucose metabolism in thyroidectomized rats. l-thyroxine therapy restored the glucose intolerance observed in radioactive iodine-treated thyroidectomized rats.