Abstract Body: Cardiovascular disease remains one of the leading causes of mortality and morbidity in the United States. Despite advancements in diagnosis and treatment, the outcomes, survival rates, and prognosis for heart failure patients continue to be poor. Recently, various therapeutic agents for post-myocardial infarction (MI) repair have been explored, including non-coding RNAs (ncRNAs). Circular RNAs (circRNAs) have emerged as a novel class of regulatory ncRNAs, functioning as microRNA (miRNA) or RNA-binding protein sponges, or by directly interacting with proteins. Using unbiased RNA sequencing on sham and MI hearts three days post-infarction, we identified differentially expressed circRNAs in post-MI hearts. Through divergent primers and quantitative real-time PCR analysis, we confirmed that several circRNAs showed altered expression between sham and MI hearts at day three. Notably, we identified a circRNA, which we named myocardial infarction-induced circular RNA (MI-circRNA), that was significantly upregulated in MI hearts compared to sham controls. To investigate the effects of MI-circRNA overexpression in the heart, we generated AAV9 viral particles and used a cardiac-specific cTnT promoter. Overexpression of MI-circRNA in post-MI hearts resulted in reduced left ventricular (LV) function. Further studies revealed that MI-circRNA overexpression disrupted calcium transients and induced cell death in HL-1 cells and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). To explore the underlying molecular mechanisms, we performed pulldown of MI-circRNA by anti-sense oligo (ASO) in healthy heart following by transcriptomics analysis. This analysis identified numerous differentially expressed protein coding genes. We selected splicing factor, Muscleblind-like-3 (MBNL3) as a potential mediator of cardiomyocyte dysfunction. These findings suggest that MI-circRNA may serve as a novel therapeutic target to prevent heart failure progression. Modulating MI-circRNA expression could represent a promising strategy for promoting cardiac repair following myocardial infarction.