Myocardial infarction (MI) triggers a significant injury response that eventually leads to adverse cardiac remodeling and heart failure, with the extracellular matrix, including collagen, plays a crucial role in this process. However, the relationship between 5-methylcytosine (m5C) modification and cardiac remodeling after MI remains unclear. In this study, an MI model was established in mice through ligation of the left anterior descending coronary artery, and hypoxia-induced primary neonatal cardiac fibroblasts were used as a cell model. Various techniques, including bioinformatics, immunofluorescence, histopathology, Western blot, and in vivo adeno-associated virus (AAV) infection, were employed to investigate the role of Alyref in cardiac remodeling following MI. We found that the expression of the m5C reader Alyref was increased in infarcted myocardial tissue in mice. Single-cell sequencing data revealed that Alyref was most significantly expressed in activated cardiac fibroblasts after MI and was involved in regulating cardiac remodeling. RNA immunoprecipitation sequencing (RIP-seq) analysis indicated that Alyref modulates the synthesis of extracellular matrix proteins, including collagen and elastin, in cardiac fibroblasts. In hypoxia-induced primary cardiac fibroblasts, siRNA-mediated Alyref knockdown reduced the synthesis of Col1a2, Col3a1, and Eln, mechanistically linked to the inhibition of the Fbln1/Loxl1 pathway. Additionally, Alyref knockdown suppressed the proliferation and transdifferentiation of cardiac fibroblasts. In vivo, AAV-mediated Alyref silencing attenuated collagen/elastin synthesis, impairing cardiac remodeling and worsening cardiac function after MI in mice. Overall, our findings demonstrate that the m5C reader Alyref regulates extracellular matrix protein synthesis in cardiac fibroblasts and represents a potential therapeutic target for modulating cardiac remodeling after MI.