Cryopreservation is a useful tool for genetic improvement which has been applied to several bivalve mollusk species. It would allow to keep the gametes of selected individuals and to preserve ex-situ the biodiversity of wild populations of the black-lip pearl oyster Pinctada margaritifera threatened of standardization by the seed transfers between the islands in French Polynesia. The ability to cryopreserve spermatozoa would bring about significant benefits to the cultured black pearl industry. Sperm freezing supposes the control of different steps: preparation of breeders, sperm collection, evaluation of sperm quality and the freezing process itself. Broodstock conditioning in hatchery allows to obtain gametes from the pearl oyster. Sperm quality is evaluated using a motility index and bioluminescence ATP measurements of spermatozoa. The following cryoprotectant additives (CPA) mixture were evaluated : (1) 0.41 M trehalose and 0, 0.45, 0.91, 1.36 M dimethyl sulfoxide (Me2SO), (2) 0.7 M trehalose and 0.8 M Me2SO and (3) 0.10 M trehalose. Freezing was made in two steps, in nitrogen vapor followed by direct plunging of spermatozoa in liquid nitrogen. Total motility was best retained when spermatozoa were cryopreserved with 0.41 M trehalose and 0, 0.45, 0.91, 1.36 M Me2SO and 0.7 M trehalose and 0.8 M Me2SO. Bioluminescence ATP measurements showed that spermatozoa conserved their energetic stock. Moreover, fertilization efficiency of thawed sperm was demonstrated using artificially matured oocytes, which allowed the development of embryos and D larvae.