Acute myocardial infarction (AMI) is serious disease with high morbidity and mortality worldwide. CD38 is an important metabolic enzyme and plays an important role in a variety of diseases. Our previous studies demonstrated that CD38 deficiency significantly reduced Ang-II-induced ventricular hypertrophy and cardiac ischemia-reperfusion injury. However, the roles of cardiomyocytic CD38 in acute myocardial infarction (AMI) remain unknown. Here, we reported that cardiomyocyte-specific CD38 deficiency (CD38(CKO)) significantly improved heart functions in AMI. We observed that CD38(CKO) remarkably reduced the fibrosis at the peri-infarct area, and inhibited the apoptosis of cardiomyocytes in infarcted area by elevating the ratio of mitochondrial Bcl2/Bax expression and increased the expressions of the mitochondrial fusion proteins Mfn1 and Mfn2 in the early stage of AMI. Consistently, knockdown of CD38 protected hypoxia-induced apoptosis in cardiomyocytes by increasing the ratio of Bcl2/Bax expression and decreasing cleaved caspase-3. More importantly, 3-TYP, a Sirt3 inhibitor, significantly increased hypoxia-induced apoptosis in CD38-deficient primary cardiomyocytes. In conclusion, our results demonstrated that CD38(CKO) suppressed apoptosis of cardiomyocytes in the infracted area of heart via activating NAD(+)/Sirt3-mediated signaling pathways.