Background- Circulating microRNAs ( miRNAs) are emerging as novel disease biomarkers. We aimed to explore the association between circulating miRNAs and the occurrence of acute myocardial infarction (AMI) in Chinese populations. Methods and Results- In the discovery stage, the plasma of 20 patients with AMI and 20 controls were pooled respectively and profiled by massively parallel sequencing. Seventy-seven miRNAs showed differential expression. Selected miRNAs were validated in 178 patients with AMI and 198 controls using quantitative reverse transcriptase polymerase chain reaction assays and further replicated in 150 patients with AMI and 150 controls. Results suggest that miR-320b and miR-125b levels were significantly lower in patients with AMI than in controls in both validation populations (P < 0.0001). Lower levels of miR-320b and miR-125b were associated with increased occurrence of AMI (adjusted odds ratio, 4.71; 95% confidence interval, 2.96-7.48 and odds ratio, 4.27; 95% confidence interval, 2.84-6.41, respectively). Addition of the 2 miRNAs to traditional risk factors led to a significant improvement in the area under the curve from 0.822 (95% confidence interval, 0.787-0.856) to 0.871 (95% confidence interval, 0.842-0.900), with a net reclassification improvement of 20.45% (P < 0.0001) and an integrated discrimination improvement of 0.16 (P < 0.0001) for patients with AMI. A functional study showed that miR-320b and miR-125b could regulate the expression profiles of genes enriched in several signal transduction pathways critical for coronary heart disease in human vascular endothelial cells. Conclusions- The plasma levels of miR-320b and miR-125b were significantly lower in patients with AMI when compared with controls, and these miRNAs may be involved in the pathogenesis of coronary heart disease. Supplemental Digital Content is available in the text.