In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411 (MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers.