Neddylation is essential for cardiomyocyte survival in the presence of oxidative stress, and it participates in autophagy regulation. However, whether MLN4924-an inhibitor of neddylation-exerts cardioprotective effects against myocardial ischemia/reperfusion (MI/R) remains unknown. In the present study, MLN4924 exerted strong cardioprotective effects, demonstrated by significantly elevated cell viability, a decreased LDH leakage rate, and improved cell morphology following H₂O₂-induced injury in vitro. MLN4924 also markedly decreased the serum myocardial zymogram level, ameliorated cardiac histopathological alterations, and alleviated left ventricular contractile dysfunction, thus limiting the cardiac infarct size in vivo compared with those in MI/R mice. Amazingly, such action of MLN4924 was abrogated by a combined treatment with the autophagic flux inhibitor, chloroquine. The mRFP-GFP-LC3 assay illustrated that MLN4924 restored the defective autophagic flux via enhancing the autolysosome formation. Notably, the expression levels of Rab7 and Atg5 were markedly up-regulated in MLN4924 treated cells and mice subjected to H₂O₂ or MI/R, respectively, while knockdown of Sirt1 in cells and heart tissue largely blocked such effect and induced autophagosome accumulation by inhibiting its fusion with lysosomes. Transmission electron microscopic analysis, histopathological assay and TUNEL detection of the heart tissues showed that the absence of Sirt1 blocked the cardioprotective effect of MLN4924 by further exacerbating the impaired autophagic flux during MI/R injury in vivo. Taken together, MLN4924 exhibited the strong cardioprotective action via restoring the impaired autophagic flux in H₂O₂-induced injury in vitro and in MI/R mice. Our work implicated that Sirt1 played a critical role in autophagosome clearance, likely through up-regulating Rab7 in MI/R.