Pearl oyster shell consists of two layers, the prismatic layer at the outside and the nacreous layer at the inside. Both layers are made of calcium carbonate, but the crystal polymorph is different; the former is made of calcite, while the latter is made of aragonite. We identified many matrix peptides and proteins from the shell of the Japanese pearl oyster (Pinctada fucata); Prismalin 14 from the prismatic layer, Pif 80/Pif 97 from the nacreous layer, LICP, LMP, and PfTIMP from the ligament, and PPP-10 from the periostracum. Among them, Pif 80 was isolated as a matrix protein that binds more selectively to aragonite than to calcite in vitro, suggesting that Pif 80 induces aragonite. Pif 97 and Pif 80 are formed from one precursor protein, and Pif 97 contains vWA domain and chitin-binding domain. Pif 80/Pif 97 containing fraction bound on the chitin film induced aragonite in vitro just like in vivo. Knockdown of the Pif 80 gene inhibited the shell growth, indicating the association of Pif 80 with pearl layer growth. Pif 80-like molecules were also identified from the black-lip pearl oyster, white-lip pearl oyster, but not in the winged pearl oyster or blue mussel. Based on the data about Pif 80/Pif 97, molecular evolution of Pif was considered. Chitin was first identified in the prismatic layer and a cDNA encoding chitin synthase was cloned.