To assess the roles of dietary protein (Pr) and calcium (Ca) level associated with excessive fluoride (F) intake and the impact of dietary Pr, Ca, and F on thyroid function, 144 30-day-old Wistar albino rats were randomly allotted to six groups of 24 (female:male = 1:1). The six groups were fed (1) a normal control (NC) diet (17.92% Pr, 0.85% Ca = NC group); (2) the NC diet and high F (338 mg NaF [=150 mg F ion]/L in their drinking water = NC+F group); (3) low Pr and low Ca diet (10.01% Pr, 0.24% Ca = LPrLCa group); (4) low Pr and low Ca diet plus high F = LPrLCa+F group; (5) high Pr and low Ca diet plus high F (25.52% Pr, 0.25% Ca = HPrLCa+F group); and (6) low Pr and high Ca diet plus high F (10.60% Pr, 1.93% Ca = LPrHCa+F group). The areas of thyroid follicles were determined by Image-Proplus 5.1, and triiodothyronine (T3), free T3 (FT3), thyroxine (T4), and free T4 (FT4) levels in serum were measured by radioimmunoassay. The histopathological study revealed obviously flatted follicular epithelia cells and hyperplastic nodules, consisting of thyroid parafollicular cells that appeared by excessive F ingestion, on the 120th day. Pr or Ca supplementation reverses the F-induced damage in malnutrition. The serum T3, FT3, T4, and FT4 levels in the NC+F group were significantly decreased and significantly increased in the LPrLCa+F group. Thus, excessive F administration induces thyroid dysfunction in rats; dietary Pr and Ca level play key roles in F-induced thyroid dysfunction.