Pearl color is affected by the nacre color of shells in Hyriopsis cumingii, and is the primary indicator of its value. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in many biological processes, including pigmentation. In this study, we used a luciferase reporter assay to identify that miR-4504 can interact with the 3'-untranslated region of the MITF gene in H. cumingii (HcMitf). After injecting mussels with the miR-4504 antagomir, the expression of miR-4504 was inhibited. Upon miR-4505 silencing, the expression of HcMitf and its downstream gene, HcTyr, were simultaneously increased. Tyrosinase activity and melanin content were also increased. The collective findings indicated that miR-4504 was involved in melanin synthesis in H. cumingii. These findings also improve our understanding of the molecular mechanisms of nacre color formation in H. cumingii. (C) 2019 Elsevier Inc. All rights reserved.