We had previously found that ATP synthase β subunit (ATPβ) plays a crucial role in regulating ATP synthesis in pancreatic β cells.This study aimed to investigate whether modulation of ATPβ expression increased hepatic ATP content and ameliorated hyperglycemia of db/db mice.ATPβ was overexpressed in the liver of db/db mice via tail injection of adenoviruses expressing ATPβ (Ad-GFP as control).7 days after viral injection,hyperglycemia, glucose intolerance and steatosis of db/db mice were markedly improved with elevated hepatic ATP content.ATPβ overexpression significantly activated Akt, and repressed gluconeogenic genes PEPCK and G6P in db/db mouse liver.In vitro, ATPβ overxpression increased ATP content in the cell and medium of cultured HepG2 cells.ATPβ overxpression activated PI3K-Akt signaling axis independent of insulin and protected against pahnitate-induced insulin resistance in HepG2 cells.ATPβ-induced activation of Akt was attenuated by ATP receptor (P2 receptor).Moreover, ATPβ overexpression also ameliorated hyperglycemia of STZ-induced type 1 diabetic mice.In conclusion, our study demonstrated that (1) Specific overexpression of ATPβ in the liver improved hyperglycemia of type 1 and type 2 diabetic mice;(2) ATPβ-mediated increase in ATP content activated PI3K-Akt signaling pathway through P2 receptor.