MicroRNAs (miRNAs) are small non-coding RNAs, which inhibit the stability and/or translation of a mRNA. miRNAs have been found to play a powerful role in various cardiovascular diseases. Recently, we have demonstrated that a microRNA (miR-181c) can be encoded in the nucleus, processed to the mature form in the cytosol, translocated into the mitochondria, and ultimately can regulate mitochondrial gene expression. However the in vivo impact of miR-181c is unknown. Here we report an in-vivo method for administration of miR-181c in rats, which leads to reduced exercise capacity and signs of heart failure, by targeting the 3′-end of mt-COX1 (cytochrome c oxidase subunit 1). We cloned miR-181c and packaged it in lipid-based nanoparticles for systemic delivery. The plasmid DNA complexed nanovector shows no apparent toxicity. We find that the mRNA levels of mitochondrial complex IV genes in the heart, but not any other mitochondrial genes, are significantly altered with miR-181c overexpression, suggesting selective mitochondrial complex IV remodeling due to miR- 181c targeting mt-COX1. Isolated heart mitochondrial studies showed significantly altered O2-consumption, ROS production, matrix calcium, and mitochondrial membrane potential in miR-181c-treated animals. For the first time, this study shows that miRNA delivered to the heart in-vivo can lead to cardiac dysfunction by regulating mitochondrial genes.