Abstract: miRNAs act as the negative regulator involved in shell formation by targeting to the 3′ UTR of the target gene. In this study, we obtained the precursor sequence of Pfm-miR-9b-5p based on the miRNA mature sequence of Pinctada fucata martensii identified before and its genome data. The precursor Pfm-miR-9b-5p had a length of 99 bp, which containing a characteristic hairpin structure. Sequence alignment showed that both the mature sequence and the precursor were highly conserved with other species. Phylogenetic analysis indicated that the precursor sequence of Pfm-miR-9b-5p clustered with mollusk and highly related to Lottia gigantean. Differential expression analysis in tissues showed that Pfm-miR-9b-5p highly expressed in mantle edge and mantle pallial which is mainly responsible for shell formation. The prediction of target genes of Pfm-miR-9b-5p showed that shell matrix protein PNU9 is one of the potential target genes of Pfm-miR-9b-5p. When Pfm-miR-9b-5p was overexpressed by injection of mimics, the expression of target gene PNU9 was significantly suppressed and the nacre layer and prismatic layer grew abnormally. Therefore, we proposed that Pfm-miR-9b-5p participated in shell formation by negatively regulating the expression of biomineralization gene PNU9 in Pinctada fucata martensii and this research provided some data to better understand the mechanisms of shell formation.