BACKGROUND: Males and females exhibit distinct anatomic and functional characteristics of the heart, predisposing them to specific disease states. METHODS: We identified microRNAs (miRNAs/miR) with sex-differential expression in mouse hearts. RESULTS: Four conserved miRNAs are present in a single locus on the X-chromosome and are expressed at higher levels in females than males. We show miRNA, miR-871, is responsible for decreased expression of the protein SRL (sarcalumenin) in females. SRL is involved in calcium signaling, and we show it contributes to differences in electrophysiology between males and females. miR-871 overexpression mimics the effects of the cardiac physiology of conditional cardiomyocyte-specific Srl-null mice. Inhibiting miR-871 with an antagomir in females shortened ventricular repolarization. The human orthologue of miR-871, miR-888, coevolved with the SRL 3' untranslated region and regulates human SRL. CONCLUSIONS: These data highlight the importance of sex-differential miRNA mechanisms in mediating sex-specific functions and their potential relevance to human cardiac diseases.