C.RF-Tshr ^hyt/hyt mice have a mutated thyroid stimulating hormone receptor (TSHR) and without thyroid hormone supplementation, these mice develop severe hypothyroidism. When hypothyroid Tshr ^hyt/hyt mice were exposed to cold (4 °), rectal temperature rapidly dropped to 23.9 ± 0.40 ° at 90 min, whereas the wild mice temperatures were 37.0 ± 0.15 °. When we carried out functional rat TSHR gene transfer into the brown adipose tissues by plasmid injection combined with electroporation, there was no effect on the serum levels of T4; although rectal temperature of the mice transfected with pcDNA3.1/Zeo-rat TSHR 90 min after cold exposure remained at 34.6 ± 0.34 °, which was significantly higher than that of Tshr hyt/hyt mice. Transfection of TSHR cDNA increased messenger RNA and protein levels of uncoupling protein-1 (UCP-1) in brown adipose tissues, and weight ratio of brown adipose tissue to overall body weight also increased. Exogenous thyroid hormone supplementation to Tshr ^hyt/hyt mice restored rectal temperature 90 min after exposure to cold (36.8 ± 0.10 °). These results indicate that not only thyroid hormone but also TSH/TSHR are involved in the expression mechanism of UCP-1 in mouse brown adipose tissue. TSH stimulates thermogenesis and functions to protect a further decrease in body temperature in the hypothyroid state.