The objective of present study was to investigate the combination effect of 6 % of glycerol and skim milk on spermatozoa motility of giant grouper, Epinephelus lanceolatus (Bloch 1970) cryopreserved for 48 hours. The ejaculates were diluted with the solvent (fish ringer extender+6 % glycerol+skim milk), the dilution ratio between sperm and diluent solution is 1:9. Samples were then equilibrated for 10 minutes at 4 °C and were frozen for 48 hours at -20 °C. Thawing was carried out for 30 seconds at 45 °C. Data were analyzed using by Anova and Tukey test. According to result ANOVA test, combination of 6 % glycerol with skim milk in spermatozoa motility after frozen had a significant effect (P > 0.05). Further, based on Tukey test, 15 %, 20 %, and 25 % of skim milk was demonstrated significantly different with control and the highest spermatozoa motility was 20 % (80.51±3.46 %).