Myocardial ischemia-reperfusion injury (MIRI) is a life-threatening complication of myocardial infarcts, with inner mitochondrial membrane protein dysfunction involved in MIRI-induced heart injury. The role of outer mitochondrial membrane protein MAVS is unknown. Here we show that MAVS expression increased in infarcted myocardium of wild-type (WT) mice. Global MAVS-knock-out (MAVS-KO) or myocardial-specific MAVS knockdown protects mice from acute and chronic MIRI, reducing apoptosis, leukocyte infiltration, etc. MIRI induces double-stranded RNA in affected myocardium, activating intracellular retinoic acid-inducible gene I (RIG-I) signaling, which led to MAVS aggregation for further non-canonical downstream signaling. MAVS aggregates recruit tumor necrosis factor associated factor family 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1), activating mitogen-activated protein kinase (MAPK) pathway and apoptosis. MAVS-KO reduces -jun-NH2 terminal kinase (JNK) phosphorylation and apoptosis. JNK inhibition benefits WT mice and JNK agonist impairs protection in MAVS-KO mice. MIRI activates RIG-I/MAVS via TAK1/TRAF6-MAPK/JNK axis, suggesting new MIRI therapeutic targets to treat MIRI.