Background The shell of bivalve mollusks is a calcified hard tissue composed of two polymorphs of CaCO3, aragonite or calcite, embedded in an organic matrix. Since the organic matrix is playing a key role in the shell formation, understanding the molecular composition of this organic framework is of great interest. The low amount of genomic data available for bivalve's species is a limit in the understanding of the dynamic process of biomineralization underlying shell formation. To increase the genomic resources and identify shell matrix proteins implicated in the biomineralization process in the pearl-producing bivalve P. margaritifera, an ESTs library from the calcifying mantle was sequenced. Results We report here the analysis of 276.738 sequences, leading to the constitution of a P. margaritifera mantle proteins catalogue of 182 sequences, potentially implicated in the biomineralization process. In situ hybridization analysis allowed the localization of pmarg-aspein and pmarg-pearlin transcripts from P. margaritifera in two distinct areas of the outer mantle epithelium. pmarg-aspein is expressed in the ventral zone, whereas pmarg-pearlin is expressed in the dorsal zone, respectively implicated in the biomineralization of calcite and aragonite. Combining the ESTs approach to a proteomic mass spectrometry analysis of proteins isolated from shell matrix proteins lead to the identification of 13 P. margaritifera shell proteins, providing direct evidence of their implication in the biomineralization process. Conclusions This ESTs study of P. margaritifera is the first description of an high-throughput pyrosequencing program generated in a mollusk species. These data generated represents a major breakthrough in the field of molluskan biomineralization.