The dusky grouper, Epinephelus marginatus, is a protogynous monandric hermaphrodite species, since individuals mature firstly as females and later as males. This makes capture and maintenance of males in captivity difficult due to their large size and older age. Thus, the use of techniques that allow controlling sex, obtaining smaller and younger males, as well as cryopreservation techniques to improve sperm availability, may contribute to improve dusky grouper reproduction in captivity. In the present study, immature fish and young females were sex reversed using 2.5 mg/kg BW 17α-methyltestosterone (α-MT) in silastic implants, two months prior gamete extraction. In all individuals a single dose of Lucrin Depot® (20 μg/kg BW, GnRHa) was enough to induce and/or increase sperm production. Volume of sperm, cell concentration, osmolarity, sperm production and motility were registered. Sperm was cryopreserved in 0.5 ml straws using 1% NaCl + 10% DMSO + 10 mg/ml BSA as extender. Post-thawed sperm quality was analysed in terms of motility, viability and fertilization. The sperm volume collected ranged from 5 to 400 μl, cell density from 1.2 to 16.3 × 109 spz/ml and sperm production from 0.04 to 3.9 × 109 spermatozoa. The percentage of motile cells at 15 s post-activation varied from 25% to 93%. Cell viability decreased in post-thawed samples (22.5%) as well as the percentage of motile cells (36.8%). However, sperm velocity (VCL and VSL) and movement pattern (Lin) were not significantly affected by cryopreservation and spermatozoa were able to fertilize the oocytes without a decrease in the fertilization rate. Fertilization rates ranged in thawed samples from 35.9% to 65.1% and only one sample was significantly different from the control (69.5%). Embryo development was impaired in some fertilization trails, registering a significant decrease in the percentage of embryos at G stage when compared with the control. This suggests that the quality of samples may be the principal requirement for improving fertilization rather than the optimization of the cryopreservation protocol. The present study demonstrated that the protocol used for sperm cryopreservation can be successfully used in sex-reversed males for the establishment of a sperm bank. This technique would contribute to the reproduction of this species benefiting production, sea-ranching and species conservation.