To elucidate the functions of α₂-adrenoceptor subtypes in metabolic regulation, we determined plasma glucose and insulin levels and tissue uptake of the glucose analogue 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) in C57Bl/6J wild-type (WT) and α_2A-adrenoceptor knockout (α_2A-KO) mice at baseline and following α₂-adrenoceptor agonist ((+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole (dexmedetomidine)) and antagonist (4-[2-ethyl-2,3-dihydro-1H-inden-2-yl]-1H-imidazole (atipamezole)) administration. Basal glucose levels were 30% lower in α_2A-KO mice than in WT mice. In WT mice, dexmedetomidine lowered insulin and elevated glucose levels, and atipamezole reduced glucose levels. In α_2A-KO mice, neither drug affected the glucose or insulin levels. [¹⁸F]FDG uptake was investigated in plasma, heart, liver, kidney, pancreas, lung, fat, and skeletal muscle. Cardiac [¹⁸F]FDG uptake was a sensitive indicator of sympathetic function. Liver [¹⁸F]FDG uptake conformed to the plasma glucose levels. In α_2A-KO mice, drug effects on [¹⁸F]FDG tissue uptake were absent. Thus, the α_2A-adrenoceptor is the α₂-adrenoceptor subtype primarily involved in the regulation of blood glucose homeostasis in vivo.