The grouper, Epinephelus lanceolatus, is a vulnerable species of high economic value. An effective protocol was developed for the cryopreservation of E. lanceolatus by comparing different extenders produced by mixing various cryoprotectants (dimethyl sulfoxide, methanol and glycerol) and diluents (MPRS, TS-2, TS-19, Cortland and Hank's). Using computer-assisted sperm analysis (CASA) and morphological analysis, the sperm motility and fertilization rates from post-thaw sperm were comparable to untreated controls. The results revealed that MPRS (containing 12% DMSO) or TS-19 (containing 12% DMSO), were the optimum extenders for protecting the sperm from cryo-damage in liquid nitrogen. The post-thaw sperm maintained high motility (90.61 ± 3.03%) and a fertilization rate (92.27 ± 2.43%) similar (P > 0.05) to fresh sperm (94.34 ± 4% and 94.10 ± 1.87%). This study is the first to report on the successful sperm cryopreservation of E. lanceolatus and provides an important tool for repopulating this species through aquaculture.