<p>Myocardial ischemia-reperfusion injury (MIRI) seriously affects myocardial function, with circular RNA’s mechanism in MIRI under exploration. This study seeks to elucidate the clinical value and function of circRNA_0007444 in MIRI. The levels of circRNA_0007444 in the serum of 94 non-MIRI patients and 80 MIRI patients were compared. The clinical significance of circRNA_0007444 was evaluated from the perspectives of differentiating MIRI patients and identifying independent risk factors for MIRI. In vitro experiments, a hypoxia/reoxygenation (H/R)-induced human cardiomyocytes AC16 cell model was used to evaluate the regulatory effects of circRNA_0007444 on cell viability, apoptosis, myocardial injury and inflammation. In mechanism, the downstream miRNAs and target genes of circRNA_0007444 were predicted, and the participation of miR-16-5p and MFN2 was evaluated through the recovery experiment. Decreasing circRNA_0007444 could discriminate MIRI patients and severs as an independent risk factor for MIRI onset. CircRNA_0007444 was negatively correlated with myocardial injury indicators. In AC16 cells, H/R induced significant downregulation of circRNA_0007444, suppressed cell viability, promoted cell apoptosis, enhanced inflammation, and induced myocardial injury. Promoting circRNA_0007444 could protect AC16 cells from H/R-induced injury. CircRNA_0007444 could target and negatively regulate miR-16-5p. miR-16-5p could target and negatively regulate MFN2. The overexpression of miR-16-5p could reverse the protective effect of circRNA_0007444 overexpressing, while overexpression of MFN2 could eliminate this effect. circRNA_0007444 can be used as a biomarker for the early screening and identification of independent risk factors of MIRI. Promoting circRNA_0007444 could alleviate H/R-induced AC16 cell injury through the miR-16-5p/MFN2 axis.</p> Graphical Abstract <p></p>