Abstract: The winged pearl oyster Pteria penguin (Röding, 1798) is an important marine cultured species that can produce high-quality seawater pearl. Melanin is the main pigment in P. penguin and largely affects the color and value of the pearl. This study analyzed the toxicity, function and signal pathway of valproic acid (VPA). Toxicity detection showed that 100 mmol/L VPA significantly reduced the survival rate of experimental animals in a time-dependent manner, and 10 mmol/L VPA for 72h was identified as the maximal safe concentration and working time for P. penguin. The liquid chromatograph-tandem mass spectrometer (LC-MS/MS) analysis showed that the content of PDCA (pyrrole-2, 3-dicarboxylic acid) and PTCA (pyrrole-2, 3, 5-tricarboxylic acid), main alkaline oxidation products of melanin, increased significantly by 43.7% (P<0.05) and 47.1% (P<0.05) after 10 and 100 mmol/L VPA for 72h. Tyrosinase activity, a marker of melanin synthesis, increased by 59.7% (P<0.05) and 136.1% (P<0.01), correspondingly. These results indicated that valproic acid can effectively accelerate the melanin synthesis in P. penguin. The qRT-PCR (quantitative real-time PCR) showed that valproic acid increased the expression of genes in melanin synthesis pathway, including Mitf (microphthalmia-associated transcription factor), Tyr (Tyrosinase), PpBcl2 (B-cell lymphoma 2) and Cdk2 (cyclin-dependent kinase 2), but had no effect on PpCreb2 (cyclin-dependent kinase 2). This suggest that valproic acid can enhance melanin synthesis of P. penguin via Mitf-Tyr-melanin pathway.