ATP synthase β subunit (ATPSβ) had been previously shown to play an important role in controlling ATP synthesis in pancreatic β-cells. This study aimed to investigate the role of ATPSβ in regulation of hepatic ATP content and glucose metabolism in diabetic mice. ATPSβ expression and ATP content were both reduced in the livers of type 1 and type 2 diabetic mice. Hepatic overexpression of ATPSβ elevated cellular ATP content and ameliorated hyperglycemia of streptozocin-induced diabetic mice and db/db mice. ATPSβ overexpression increased phosphorylated Akt (pAkt) levels and reduced PEPCK and G6pase expression levels in the livers. Consistently, ATPSβ overexpression repressed hepatic glucose production in db/db mice. In cultured hepatocytes, ATPSβ overexpression increased intracellular and extracellular ATP content, elevated the cytosolic free calcium level, and activated Akt independent of insulin. The ATPSβ-induced increase in cytosolic free calcium and pAkt levels was attenuated by inhibition of P2 receptors. Notably, inhibition of calmodulin (CaM) completely abolished ATPSβ-induced Akt activation in liver cells. Inhibition of P2 receptors or CaM blocked ATPSβ-induced nuclear exclusion of forkhead box O1 in liver cells. In conclusion, a decrease in hepatic ATPSβ expression in the liver, leading to the attenuation of ATP-P2 receptor-CaM-Akt pathway, may play an important role in the progression of diabetes.