The process of freezing and thawing of semen could lead spermatozoa death and low fertility for alive spermatozoa. This research was subjected to determine the optimum concentration of vitamin C in diluent media to improve the quality of non-sexing and sexing result of thawed dairy cattle (Bos taurus) spermatozoa. The method used was completely randomized design with 3x4 factorial consisting of spermatozoa isolation and vitamin C concentration. Spermatozoa isolation factors were non-sexing and sexing results (X and Y spermatozoa). Vitamin C concentrations factors were 0% (K); 0.25% (P1); 0.50% (P2) and 0.75% (P3). Data were analyzed using analysis of variance (ANOVA) and Duncan's Multiple Range Test 95%. The optimum concentration of vitamin C in diluent media to improve the quality of non-sexing thawed dairy cattle (Bos taurus) spermatozoa was 0.25%. While the optimum concentration of vitamin C in diluent medium to improve quality of thawed dairy cattle (Bos taurus) spermatozoa both X and Y was 0.50%. Administration of vitamin C in diluent media could improve the quality of thawed non-sexed and sexed (X and Y spermatozoa) spermatozoa in dairy cattle (Bos taurus).Kata Kunci: Spermatozoa, Bos taurus, Vitamin C, Sexing, Diluent, Thawing