Nile tilapia, Oreochromis niloticus, is one of the most important cultivated species in freshwater aquaculture due to its fast growth rate. microRNAs (miRNAs) are approximately 22-nt noncoding RNAs that have emerged as key regulators of skeletal muscle development, but the knowledge of the identity of myogenic miRNAs and their targets in tilapia remains limited. Here, we identify 25 conserved miRNAs in tilapia skeletal muscle using small RNA cloning. Among these miRNAs, miR-1, miR-27a, miR-133a, and miR-206 are differentially expressed in different development stages. Antagomir administration results in a profound decrease in endogenous expression of miR-1, miR-133a, miR-27a or miR-206, while the expression of putative target genes, including HDAC4, SRF, Pax3 or Pax7, is found to be significantly increased. The inverse expression correlation between miRNAs and putative target genes provides evidence that miR-1, miR-133a, miR-27a or miR-206 can directly regulate HDAC4, SRF, Pax 3 or Pax 7 in vivo. The finding would shed new light on the molecular mechanism of skeletal muscle development. (C) 2012 Elsevier B.V. All rights reserved.