microRNAs (miRNAs) represent approximately 4% of the genes in vertebrates, where they regulate deadenylation, translation, and decay of the target messenger RNAs (mRNAs). The integrated role of miRNAs to regulate gene expression and cell function remains largely unknown. Therefore, to identify the targets coordinately regulated by muscle miRNAs in vivo, we performed gene expression arrays on muscle cells sorted from wild type, dicer mutants, and single miRNA knockdown embryos. Our analysis reveals that two particular miRNAs, miR-1 and miR-133, influence gene expression patterns in the zebrafish embryo where they account for >54% of the miRNA-mediated regulation in the muscle. We also found that muscle miRNA targets (1) tend to be expressed at low levels in wild-type muscle but are more highly expressed in dicer mutant muscle, and (2) are enriched for actin-related and actin-binding proteins. Loss of dicer function or down-regulation of miR-1 and miR-133 alters muscle gene expression and disrupts actin organization during sarcomere assembly. These results suggest that miR-1 and miR-133 actively shape gene expression patterns in muscle tissue, where they regulate sarcomeric actin organization.