Concentric pathological cardiac hypertrophy occurs following pressure-overload. It is proposed that preventing this hypertrophy could prevent the progression towards heart failure. Our work demonstrates that AMPK activation inhibits cardiomyocyte hypertrophy independently of the previously proposed downstream targets. By contrast, we show that the anti-hypertrophic action of AMPK is caused by the inhibition of protein O-GlcNAcylation. Our study reveal that AMPK activation induces phosphorylation and inhibition of GFAT as well as decreases OGT protein expression, both enzymes being required for O-GlcNAcylation. The AMPK-mediated inhibition of protein O-GlcNAcylation and its central role in the reduction of cardiac hypertrophy bring new potential therapeutic opportunities for the treatment of this pathology.