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The paired-box 3 (<i>Pax3</i>) is a transcription factor and it plays an important part in melanin synthesis. In this study, a new <i>Pax3</i> gene was identified from <i>Pteria penguin</i> (<i>R&#246;ding</i>, <i>1798</i>) (<i>P. penguin</i>) by RACE-PCR (rapid-amplification of cDNA ends-polymerase chain reaction) and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of <i>PpPax3</i> was 2250 bp long, containing an open reading fragment of 1365 bp encoding 455 amino acids. Amino acid alignment and phylogenetic tree showed <i>PpPax3</i> shared the highest (69.2%) identity with <i>Pax3</i> of <i>Mizuhopecten yessoensis</i>. Tissue expression profile showed that <i>PpPax3</i> had the highest expression in mantle, a nacre-formation related tissue. The <i>PpPax3</i> silencing significantly inhibited the expression of <i>PpPax3</i>, <i>PpMitf</i>, <i>PpTyr</i> and <i>PpCdk2</i>, genes involved in <i>Tyr</i>-mediated melanin synthesis, but had no effect on <i>PpCreb2</i> and an increase effect on <i>PpBcl2</i>. Furthermore, the <i>PpPax3</i> knockdown obviously decreased the tyrosinase activity, the total content of eumelanin and the proportion of PDCA (pyrrole-2,3-dicarboxylic acid) in eumelanin, consistent with influence of tyrosinase (<i>Tyr</i>) knockdown. These data indicated that <i>PpPax3</i> played an important regulating role in melanin synthesis by <i>Tyr</i> pathway in <i>P. penguin</i>.