MyoD is one of the helix-loop-helix proteins regulating muscle-specific gene expression in tilapia. Tight regulation of the MyoD protein level is necessary for the precise regulation of skeletal muscle development. MicroRNAs (miRNAs) are a class of regulatory RNAs that post-transcriptionally regulate gene expression. An increasing amount of evidence has suggested that miRNAs play an important role in regulating skeletal muscle development. We reasoned that MyoD expression may be regulated by miRNAs. Predictions from bioinformatics have identified a putative miR-203b target site in the 3'-UTR of the MyoD gene. Interestingly, miR-203b expression is negatively correlated with MyoD expression, whereas miR-203b suppression leads to a significant increase in MyoD expression, thereby activating MyoD downstream genes. A 3'-UTR luciferase reporter assay further verifies the direct interaction between miR-203b and MyoD. Taken together, our results reveal a novel molecular mechanism in which miRNA participates in transcriptional circuits that regulate gene expression in tilapia skeletal muscle.