Background <p>Acute myocardial infarction (AMI) often occurs suddenly and leads to serious consequences. Therefore, there is an urgent need to find a new biomarker for AMI.</p> Methods <p>MiR-1299 expression was measured by RT-qPCR. The diagnostic value of miR-1299 in AMI was measured by the receiver operating characteristic (ROC) curve. Chi-squared test was utilized to analyze associations between the clinicopathological characteristics of AMI patients and miR-1299 levels. MiR-1299 was overexpressed or inhibited by transfection in hypoxic H9c2 cells. Then, cell activity and the expression of myocardial injury markers in H9c2 cells were detected.</p> Results <p>MiR-1299 was increased in AMI and had a high diagnostic value in AMI. In AMI, the expression level of miR-1299 is closely related to WBC and current smoking. In hypoxic H9c2 cells, the overexpression of miR-1299 reduced the cell viability, while the inhibition of miR-1299 increased the cell viability. In addition, the overexpression of miR-1299 increased cTnⅠ, CK-MB, and MYO, while the inhibition of miR-1299 significantly reduced cTnⅠ, CK-MB, and MYO in hypoxic H9c2 cells.</p> Conclusion <p>MiR-1299 was increased in AMI and had a high diagnostic value. In hypoxic H9c2 cells, miR-1299 regulated proliferation and the expression of myocardial injury markers, suggesting that miR-1299 may be a potential biomarker of AMI.</p>