Background Black pearl farming is based on culture of the blacklip pearl oyster Pinctada margaritifera (Mollusca, lophotrochozoa), a protandrous hermaphrodite species. At first maturation, all individuals are males. The female sex appears progressively from two years old, which represents a limitation for broodstock conditioning for aquaculture production. In marine mollusks displaying hermaphroditic features, data on sexual determinism and differentiation, including the molecular sex determining cascade, are scarce. To increase genomic resources and identify the molecular mechanisms whereby gene expression may act in the sexual dimorphism of P. margaritifera, we performed gonad transcriptome analysis. Results The gonad transcriptome of P. margaritifera was sequenced from several gonadic samples of males and females at different development stages, using a Next-Generation-Sequencing method and RNAseq technology. After Illumina sequencing, assembly and annotation, we obtained 70,147 contigs of which 62.2% shared homologies with existing protein sequences, and 9% showed functional annotation with Gene Ontology terms. Differential expression analysis identified 1,993 differentially expressed contigs between the different categories of gonads. Clustering methods of samples revealed that the sex explained most of the variation in gonad gene expression. K-means clustering of differentially expressed contigs showed 815 and 574 contigs were more expressed in male and female gonads, respectively. The analysis of these contigs revealed the presence of known specific genes coding for proteins involved in sex determinism and/or differentiation, such as dmrt and fem-1 like for males, or foxl2 and vitellogenin for females. The specific gene expression profiles of pmarg-fem1-like, pmarg-dmrt and pmarg-foxl2 in different reproductive stages (undetermined, sexual inversion and regression) suggest that these three genes are potentially involved in the sperm-oocyte switch in P. margaritifera. Conclusions The study provides a new transcriptomic tool to study reproduction in hermaphroditic marine mollusks. It identifies sex differentiation and potential sex determining genes in P. margaritifera, a protandrous hermaphrodite species.