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Mollusk shell matrix proteins are important for the formation of organic frameworks, crystal nucleation, and crystal growth in  Pinctada fucata martensii  ( P .  f .  martensii ). MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in many biological processes, including shell formation. In this study, we obtained the full-length sequence of  Pif97-like  gene in  P .  f .  martensii  ( PfmPif97-like ).  PfmPif97-like  was mainly distributed in mantle pallial and mantle edge. Correlation analysis indicated that the average shell thickness and weight showed a positive correlation with  PfmPif97-like  expression ( P < 0 . 05 ). The inner surface of the nacreous layer and prismatic layer showed atypical growth when we knocked down the expression of  PfmPif97-like  by RNA interference (RNAi). We used a luciferase reporter assay to identify that miR-9b-5p of  P .  f .  martensii  (Pfm-miR-9b-5p) downregulated the expression of  PfmPif97-like  by interacting with the 3′-untranslated region (UTR) while we obtained the same result by injecting the Pfm-miR-9b-5p mimics in vivo. After injecting the mimics, we also observed abnormal growth in nacre layer and prismatic layer which is consistent with the result of RNAi. We proposed that  PfmPif97-like  regulated by Pfm-miR-9b-5p participates in shell formation of  P .  f .  martensii . These findings provide important clues about the molecular mechanisms that regulate biomineralization in  P .  f .  martensii .

PfmPif97-like regulated by Pfm-miR-9b-5p participates in shell formation in Pinctada fucata martensii