Muscle fructose 1,6-bisphosphatase (FBP2), a regulatory enzyme of glyconeogenesis, binds to mitochondria where it interacts with proteins involved in regulation of energy homeostasis. Here, we show that the quaternary structure of FBP2 plays a crucial role in this interaction, and that the AMP-driven transition of the FBP2 subunit arrangement from active to inactive precludes its association with the mitochondria. Moreover, we demonstrate that truncation of the evolutionarily conserved N-terminal residues of FBP2 results in a loss of its mitochondria-protective functions. This strengthenes the recently raised hypothesis that FBP2 evolved as a regulator of not only glycogen storage but also mitochondrial function in contracting muscle.