•We obtained the full length of TIMP gene in Pinctada martensii.•Pm-TIMP mRNA was highly expressed in the mantle and pearl sac.•RNA interference was used to elucidate the function of Pm-TIMP gene.•The obtained Pm-TIMP participated in nacre formation in Pinctada martensii. Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901bp long, containing a 5′ untranslated region (UTR) of 51bp, a 3′ UTR of 169bp, and an open reading fragment (ORF) of 681bp encoding 226 amino acids with an estimated molecular mass of 23.37kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.