Background: Previous studies showed controversies about the association of vitamin D deficiency and thyroid dysfunction and up to our knowledge there was no study that were conducted to study the effect of vitamin D deficiency on thyroid function. Metformin is an antidiabetic drug that is widely used and also there was contradictory data about its effect on thyroid function. Aim: To identify whether vitamin D deficiency is considered a cause for hypothyroidism or not. Also, to investigate the effect of metformin treatment on thyroid function in a rat model of vitamin D deficiency. Materials and Methods: 40 Adult male albino rats of local strains were divided randomly into 5 equal groups; control, control metformin treated (control+metformin), vitamin D deficient, vitamin D deficient metformin treated (vitamin D deficient+metformin) and vitamin D deficient vitamin D treated (vitamin D deficient+vitamin D). Results: Vitamin D deficiency model significantly increased final body mass index (BMI), relative thyroid weight, serum thyroid stimulating hormone (TSH), serum reverse triiodothyronine (rT3) and hepatic deiodinase 3 activity but significantly declined serum levels of each of triiodothyronine (T3), thyroxin (T4), 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)2D) when compared to control group. Metformin treatment of vitamin D deficient rats significantly reduced relative thyroid weight with a significant decreased in TSH level when compared with vitamin D deficient group. A significant negative correlation was reported between serum 25(OH) D and each of final BMI, serum TSH, relative thyroid weight, hepatic deiodinase activity and serum rT3 but it was positively correlated to serum level of both T3 and T4. Histopathology and histomorphometric studies of the thyroid gland in vitamin D deficient rats showed a large number of follicles with increased dimensions, the follicular colloid showed no vacuolation, the amount of colloid increased and the follicles were found to be lined by simple cuboidal epithelial cells with basal nuclei. Conclusion: As vitamin D deficiency decreased thyroid function through affecting hepatic deiodinase 3 activity and this change was deteriorated with vitamin D supplementation, thus, monitoring of vitamin D levels and its supplementation may play a role in management of hypothyroidism. Also, metformin exerted an anti-proliferative activity, improved thyroid structure and lowered TSH without affecting serum levels of T4 or T3, thus it could provide a rationale for an innovative therapy of thyroid proliferative diseases.