MicroRNAs are highly-conserved, non-coding RNAs involved in post-transcriptional gene silencing. MicroRNAs have been shown to be involved in a range of biological processes including myogenesis and muscle regeneration. The objective of this study was to test the hypothesis that microRNA expression is altered in dystrophic muscle, with the greatest change occurring, of the muscles examined, in the diaphragm. The expression of the muscle-enriched microRNAs was determined in the soleus, plantaris and diaphragm muscles of control and dystrophin-deficient (mdx) mice by semi-quantitative PCR. In the soleus and plantaris, expression of the mature microRNA 133a (miR-133a) and miR-206, respectively, was decreased by ~25% whereas in the diaphragm, miR-206 expression increased by 4.5-fold relative to control. The increased expression of miR-206 in the mdx diaphragm was paralleled by a 4.4-fold increase in primary microRNA-206 (pri-miRNA-206) transcript level. Expression of Myod1 was elevated 2.7-fold only in the mdx diaphragm consistent with an earlier finding demonstrating Myod1 can activate pri-miRNA-206 transcription. Transcript levels of Drosha and Dicer, major components of microRNA biogenesis pathway, were unchanged in mdx muscle suggesting the pathway is not altered under dystrophic conditions. Previous in vitro analysis found miR-206 was capable of repressing utrophin expression; however, under dystrophic conditions both utrophin transcript and protein levels were significantly increased by 69% and 3.9-fold, respectively, a finding inconsistent with microRNA regulation. These results are the first to report alterations in expression of muscle-enriched microRNAs in skeletal muscle of the mdx mouse suggesting microRNAs may have a role in the pathophysiology of muscular dystrophy.