MicroRNAs (miRNAs) are a class of non-coding RNA molecules with post-transcriptional regulatory activity in various biological processes. Pearl oyster Pinctada fucata martensii is one of the main species cultured for marine pearl production in China and Japan. In this study, we constructed two small RNA libraries of mantle central (MC) and mantle edge (ME) from P. f. martensii and obtained 24,175,537 and 21,593,898 clean reads, respectively. A total of 258 miRNAs of P. f. martensii (Pm-miRNA) were identified, and 93 differentially expressed miRNAs (DEMs) including 49 known Pm-miRNAs and 44 novel Pm-miRNAs were obtained from the MC and ME. The target transcripts of these DEMs were obviously enriched in neuroactive ligand-receptor interaction pathway, and others. After over-expression of Pm-miR-124 and Pm-miR-9a-5p in the MC by mimic injection into the muscle of P. f. martensii, nacre exhibited a disorderly growth as detected by scanning electron microscopy. Pm-nicotinic acetylcholine receptor alpha subunit, Pm-neuropeptide Y and Pm-chitin synthase were investigated as the targets of Pm-miR-124; and Pm-tumor necrosis factor receptor associated factor 2 and Pm-chitin synthase were investigated as the targets of Pm-miR-9a-5p. These predicted target transcripts were down-regulated after the over-expression of Pm-miR-124 and Pm-miR-9a-5p in MC. This study comprehensively analyzed the miRNAs in mantle tissues to enhance our understanding of the regulatory mechanism underlying shell formation.