Background: Myofilament protein phosphorylation is central to cardiac muscle contractile regulation. Results: AMP-activated protein kinase (AMPK) phosphorylates troponin I (TnI) to increase cardiac contraction and blunt the effects of TnI protein kinase A phosphorylation. Conclusion: AMPK myofilament signaling represents a novel mechanism to regulate cardiac contraction. Significance: AMPK links metabolics to TnI regulation of cardiac muscle function and adrenergic regulation.