Purpose This study sought to probe the feasibility of performing total thyroidectomy in the mouse using a nonthyroidal hNIS expressing tumor model. Materials and Methods Our thyroidectomy protocol included thorough excision of both lobes and the isthmus. For evaluating the completeness of thyroidectomy, we compared the $^{99m}Tc$-pertechnetate scans taken before and after thyroidectomy. The prostate cancer cell line was subcutaneously inoculated 2 weeks after the thyroidectomy. When the tumor reached 5-10 mm in diameter, Ad5/35-E4PSESE1a-hNIS was injected intratumorally, and $^{131}I$ scans were performed. The radioiodine uptakes of the neck and the tumor were compared with those of the other regions. Results Total thyroidectomy was performed in 13 mice. Although 38.5% died during or just after thyroidectomy, the others survived in good health for 2 months. Thyroid tissue was completely eliminated using our protocol; the residual uptake of $^{99m}Tc$-pertechnetate was minimal in the neck area. The neck/background uptake ratio after thyroidectomy was significantly lower than that before thyroidectomy (p<0.05). Non-thyroidal tumor models were successfully established in all the surviving mice. Radioiodine accumulation in the tumors was visualized on $^{131}I$ scans, and the neck uptakes were minimal. Conclusion Using our total thyroidectomy protocol, we successfully established a hNIS-transfected prostate cancer model with a minimal accumulation of radioiodine in the neck. The relatively high mortality after surgery can be a problem, and this might be reduced by minimizing the surgical stress.