Background and Objectives: N-glycosylation, a crucial post-translational modification, is well-recognized for its pivotal role in cardiovascular functions. N-acetylglucosaminyltransferase V (GnT-V) is one of the major glycosyltransferases that determine the complexity of N-glycans in N-glycosylation modification. This study aimed to explore the role of GnT-V in myocardial infarction (MI). Methods: Proteomics and N-glycoproteomic analysis were performed on myocardial tissues for the N-glycosylation profile after MI. Adeno-associated virus (AAV) with a mouse cTnT promoter was utilized to induce overexpression of GnT-V in the heart for the role of GnT-V in MI. Echocardiography and histological analysis were used to evaluate the effect of GnT-V on MI. For the potential mechanisms of GnT-V, proteomic analysis was performed on cardiomyocytes that were subjected to GnT-V overexpression and hypoxic stress. The results were validated by western blot, lectin blot and immunoprecipitation assays, and confirmed with PNGase F and tunicamycin treatment. Results: N-glycosylation of protein was significantly reduced after MI, which could be related to a decrease in the expression levels of GnT-V and its target glycans. Targeted GnT-V overexpression in the heart by using AAV improved cardiac function and reduced the infarct size after MI. Further, proteomics analysis of cardiomyocytes revealed that insulin-like growth factor-binding protein 3 (IGFBP3) was targeted by GnT-V and induced degradation through the lysosome pathway. Consequently, the insulin-like growth factor 1 receptor (IGF1R) signaling pathway was activated through overexpression of GnT-V. Conclusion: Our findings suggest that promoting the IGF1R signaling cascades by regulating the N-glycosylation of certain proteins in the signaling pathway, especially through GnT-V, may act as a promising strategy for treating MI.