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Brown adipocytes mainly utilize glucose and fatty acids to produce energy, which play key roles in thermogenesis. Furthermore, brown adipocytes also utilize other substrates, such as amino acids, for energy expenditure in various conditions. Here, we report the new physiological roles of proton-coupled amino acid transporters, SLC36A2 and SLC36A3, on global energy metabolism. The relative mRNA expression levels of both <i>Slc36a2</i> and <i>Slc36a3</i> were all highest in brown adipose tissue. We then generated global <i>Slc36a2</i> and <i>Slc36a3</i> knockout mice to investigate their functions in metabolism. Neither loss of <i>Slc36a2</i> nor <i>Slc36a3</i> affected the body weight and body composition of the mice. <i>Slc36a2</i> knockout mice exhibited increased oxygen consumption during the daytime. After cold treatment, inhibition of <i>Slc36a2</i> significantly decreased the mass of brown adipose tissue compared to wildtype mice, while it lowered the expression level of <i>Cpt1a</i>. Moreover, the serum lipid levels and liver mass were also decreased in <i>Slc36a2</i> knockout mice after cold treatment. On the contrary, <i>Slc36a3</i> knockout impaired glucose tolerance and up-regulated serum LDL-cholesterol concentration. Thus, SLC36A2 and SLC36A3 play central and different roles in the energy metabolism of the mice.