Cryopreservation is a freezing method specific for cells such as gametes and tissues in a sub-zero temperature. The role of cryoprotectant (cryoprotective agent) is essential to reduce the cryoinjury and retain the original biological function of gametes during cryopreservation. This study looked into the effectiveness of four types of cryoprotectants (dimethyl sulfoxide, sucrose, egg yolk and honey) at four concentrations (5%, 10%, 15%, and 20%) on the spermatozoa of tropical oyster, Magallana bilineata. Sperm exposed to DMSO 10% underwent freezing has recorded the highest fertilization rate, which was significantly higher than other treatments but showed no significant differences with the control (fresh sperm). The usage of sucrose is less effective compared to DMSO, but showed no significant differences with egg yolk. This study is important to determine the best cryoprotectant to be used for spermatozoa cryopreservation of M. bilineata, to provide a solution to oyster hatchery operators to secure all year-round spawning using cryopreserved sperms.