Key points MicroRNA (miRNA)‐based therapies are in development for numerous diseases, including heart disease. Currently, very limited basic information is available on the regulation of specific miRNAs in male and female hearts in settings of disease. The identification of sex‐specific miRNA signatures has implications for translation into the clinic and suggests the need for customised therapy. In the present study, we found that a miRNA‐based treatment inhibiting miRNA‐34a (miR‐34a) was more effective in females in a setting of moderate dilated cardiomyopathy than in males. Furthermore, the treatment showed little benefit for either sex in a setting of more severe dilated cardiomyopathy associated with atrial fibrillation. The results highlight the importance of understanding the effect of miRNA‐based therapies in cardiac disease settings in males and females. Abstract MicroRNA (miRNA)‐34a (miR‐34a) is elevated in the diseased heart in mice and humans. Previous studies have shown that inhibiting miR‐34a in male mice in settings of pathological cardiac hypertrophy or ischaemia protects the heart against progression to heart failure. Whether inhibition of miR‐34a protects the female heart is unknown. Furthermore, the therapeutic potential of silencing miR‐34a in settings of dilated cardiomyopathy (DCM) and atrial fibrillation (AF) has not been assessed previously. In the present study, we examined the effect of silencing miR‐34a in males and females in (1) a model of moderate DCM and (2) a model of severe DCM with AF. The cardiac disease models were administered with a locked nucleic acid‐modified oligonucleotide (LNA‐antimiR‐34a) at 6–7 weeks of age when the models display cardiac dysfunction and conduction abnormalities. Cardiac function and morphology were measured 6 weeks after treatment. In the present study, we show that inhibition of miR‐34a provides more protection in the DCM model in females than males. Disease prevention in LNA‐antimiR‐34a treated DCM female mice was characterized by attenuated heart enlargement and lung congestion, lower expression of cardiac stress genes (B‐type natriuretic peptide, collagen gene expression), less cardiac fibrosis and better cardiac function. There was no evidence of significant protection in the severe DCM and AF model in either sex. Sex‐ and treatment‐dependent regulation of miRNAs was also identified in the diseased heart, and may explain the differential response of males and females. These studies highlight the importance of examining the impact of miRNA‐based drugs in both sexes and under different disease conditions.