MyoD is one of the helix-loop-helix proteins regulating muscle-specific gene expression in tilapia. Tight regulation of 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. Increasing evidences have suggested that miRNAs play an important role in regulating skeletal muscle development. We reasoned that MyoD expression may be regulated by miRNAs. Bioinformatics prediction identify a putative miR-203b target site in the 3’-UTR of MyoD gene. Interestingly, miR-203b expression is negatively correlated is negatively correlated with MyoD expression. miR-203b suppression leads to a significant increase in MyoD expression, thereby activating MyoD downstream gene. 3’-UTR luciferase reporter assay further verifies the direct interaction between miR-203b and MyoD. Taken together, our studies reveal a novel molecular mechanism in which miRNA participates in transcriptional circuits that regulates gene expression in tilapia skeletal muscle.