Hypothyroidism, marked by low thyroid hormone production, often requires lifelong hormone replacement, which may not fully normalize function. This study compared three thyroid transplant. Fifty mature male albino rats were segregated into three cohorts: control, thyroidectomy-only, and thyroidectomy with transplantation. The transplant group had three intervention subgroups: (1) thyroid allografts alone, (2) allografts with MSCs co-transplantation, (3) engineered thyroid cell sheets. Blood samples were collected at baseline, one week pre-thyroidectomy, one week post-thyroidectomy/pre-transplant, and weekly for four weeks post-transplant for biochemical analysis. Thyroid tissues were harvested at the study endpoint for comparative histological and molecular analyses. Pre-thyroidectomy hormone levels showed no significant differences between groups one week before the thyroidectomy procedure. However, after thyroidectomy, the average serum free T3 and free T4 concentrations were meaningfully diminished, whereas the average serum TSH level was considerably elevated in the follow-up measurements. Conversely, the thyroid allograft-MSCs conjugates demonstrated superior therapeutic effects, with enhanced thyroid hormone production and improved histological structure. In contrast, the tissue-engineered thyroid cell sheets showed a more transient therapeutic effect, likely due to the regenerative capacity and differentiation of MSCs in the thyroid tissue. These findings suggest that stem cells are crucial for both tissue-engineered thyroid cell sheets and thyroid allografts for synthesizing thyroid tissue, which may be more promising approaches for treating hypothyroidism, offering potential alternatives to traditional hormone replacement therapy thyroid allograft alone, allograft-mesenchymal stem cells (MSCs) conjugates, and tissue-engineered thyroid cell sheets to assess their effects on restoring thyroid function and structure post-thyroidectomy.