Brown adipose tissue (BAT) is a primary site for non-shivering thermogenesis in mammals. In cold temperature, β-adrenergic stimulation activates mitochondrial uncoupling protein 1 (UCP1), which consequently generates heat by uncouples of the respiratory chain. Previous report suggested that intracellular Ca²⁺ signaling induced by transient receptor potential vanilloid 2 (TRPV2) activates UCP1 in BAT thermogenesis. However, molecular mechanisms of intracellular Ca²⁺ signaling in BAT are not well characterized. We have been systematically studying the functional roles of Na⁺/Ca²⁺ exchangers, which regulate intracellular Ca²⁺ signaling, using genetically altered mice and specific inhibitors. We recently found that Na⁺/Ca²⁺ exchanger type 1 (NCX1) is abundantly expressed in interscapular BAT (iBAT) from wild-type mice. Therefore, we here analyzed functional role of NCX1 in BAT thermogenesis. Intriguingly, we observed that adipose tissue-specific conditional NCX1 knockout mice, as well as heterozygous NCX1 knockout mice, did not maintain their core body temperature in cold exposure. Furthermore, UCP1 transcripts in iBAT were significantly decreased in NCX1 knockout mice compared with wild-type mice. These results suggest that NCX1 may contribute to BAT thermogenesis against cold environment.