Objectives: In this present in vivo study, it is intended to examine the effects of fluoride on metabolic functions of four discrete regions of rat brain associated with thyroidal insufficiency. Background: Fluoride contamination in drinking water is a major health issue. Adverse health effects of fluoride include dental and muscle fluorosis, lowering mental proficiency, neurological disorders and oxidative stress in soft tissues including brain. Thyroid is mainly concerned with regulation of metabolic homeostasis and thus supposed to be altered by fluoride toxicity. Rationale: Earlier observations indicate that fluoride toxicity imposes certain adverse effects on brain metabolic activities. Alteration in metabolic profile in brain may be affected by the thyroid gland as because this gland takes care of the overall metabolic integration of the body. Significance: The present study explores the detail mechanism of metabolic alteration of different parts of rat brain namely cerebrum, cerebellum, pons and medulla by fluoride, and also evaluates through a comparative analysis that which region is mostly affected by it. Furthermore, this study also suggests a link between brain metabolic profile and thyroid function. Methods: Male rats of Wistar strain (N=6) were orally fed with 20 mg/kg/day fluoride for 30 days. Results: Following fluoride exposure, total protein content depleted more significantly in cerebrum and medulla as compared with other brain regions. The proleolytic enzyme activity was severely affected by fluoride, especially in medulla. Changes in acidic, basic and neutral protein contents reveal that fluoride altered those parameters in a tissue specific manner. Nucleic acid contents were markedly reduced in medulla and pons by fluoride, whereas RNase activity increased in the respective brain regions. Protein carbonylation is pronounced in cerebral tissue. The neurotransmitter level was markedly reduced in cerebellum in comparison with others. Additionally, fluoride also altered thyroid metabolism as specified by depletion of nucleic acid contents and inhibition of the activities of thyroidal enzymes along with significant decrease in serum T3 and T4. Conclusion: It is suggested that fluoride altered metabolic homeostasis in cerebrum, cerebellum, pons, medulla in a tissue specific manner that might be correlated with thyroidal insufficiency.