MicroRNAs (miRNAs) are small, single-stranded, non-coding RNAs that function as negative regulators of genes expression. They are transcribed from endogenous DNA, and form hairpin structures (termed as pre-miRNAs) that are processed to form mature miRNAs. It remains largely unknown as to the molecular consequences of the natural genetic variation in pre-miRNAs. Here, we report that a A>G polymorphism (rs71428439) is located in Homo sapiens miR-149 stem-loop region. This polymorphism results in a change in the structure of miR-149 precursor. Our results showed that the genotype distribution of this polymorphism in myocardial infarction cases was significantly different from that in the control subjects. We examined the biological significance of this polymorphism on the production of mature miR-149, and observed that the G-allelic miR-149 precursor displayed a lower production of mature miR-149 compared with the A-allelic one. Further investigations disclosed that miR-149 could withstand mitochondrial fission and apoptosis through targeting the pro-apoptotic factor p53-upregulated modulator of apoptosis (Puma). Enforced expression of miR-149 promoted cell survival, whereas knockdown of miR-149 rendered cells to be sensitive to apoptotic stimulation. Intriguingly, the A to G variation led pre-miR-149 to elicit an attenuated effect on the inhibition of mitochondrial fission and apoptosis. Finally, this polymorphism exerts its influence on cardiac function in mouse model of myocardial infarction. These data suggest that this polymorphism in miR-149 precursor may result in important phenotypic traits of myocardial infarction. Our finding warrant further investigations on the relationship between miR-149 polymorphism and myocardial infarction.