Post-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However， ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7， a SET domain-containing lysine methyltransferase， is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. Here we present evidence that the precise activity of ATG16L1 protein in hypoxia/reoxygenation (H/R)-treated cardiomyocytes is regulated by a balanced methylation and phosphorylation switch. We first show that H/R promotes autophagy and decreases SETD7 expression， whereas autophagy inhibition by 3-MA increases SETD7 level in cardiomyocytes， implying a tight correlation between autophagy and SETD7. Then we demonstrate that SETD7 methylates ATG16L1 at lysine 151 while KDM1A/LSD1 (lysine demethylase 1A) removes this methyl mark. Furthermore， we validate that this methylation at lysine 151 impairs the binding of ATG16L1 to the ATG12-ATG5 conjugate， leading to inhibition of autophagy and increased apoptosis in H/R-treated cardiomyocytes. However， the cardiomyocytes with shRNA-knocked down SETD7 or inhibition of SETD7 activity by a small molecule chemical， display increased autophagy and decreased apoptosis following H/R treatment. Additionally， methylation at lysine 151 inhibits phosphorylation of ATG16L1 at S139 by CSNK2 which was previously shown to be critical for autophagy maintenance， and vice versa. Together， our findings define a novel modification of ATG16L1 and highlight the importance of an ATG16L1 phosphorylation-methylation switch in determining the fate of H/R-treated cardiomyocytes.