Spermatoza yang melalui proses pembekuan seringkali mengalami kerusakan karena terbentuknya kristal es. Pembentukan kristal es dapat menyebabkan penurunan kualitas spermatozoa pasca thawing terutama penurunan motilitas. Penambahan krioprotektan dalam pengencer dapat meminimalisir kerusakan sel. Salah satu jenis krioprotektan yang sering digunakan pada pembekuan semen adalah gliserol. Penelitian ini bertujuan untuk melihat pengaruh penambahan gliserol dalam pengencer Tris Kuning Telur (TKT) terhadap kualitas spermatozoa (motilitas, spermatozoa hidup, dan MPU) sapi aceh yang disimpan dalam nitrogen cair. Desain penelitian menggunakan Rancangan Acak Lengkap (RAL) yang terdiri dari 3 perlakuan dan 6 pengulangan. Pengamatan kualitas spermatozoa dilakukan setelah pembekuan di dalam nitrogen cair. Data dianalisis dengan uji Analysis of Variance (ANOVA) pola satu arah. Hasil kualitas spermatozoa pada masing-masing konsentrasi gliserol (3%, 5%, dan 7%) berturut-turut untuk hasil motilitas sebesar: 17,17±12,17%, 51,17±11,13%, dan 33,33±8,76%; hasil spermatozoa hidup sebesar: 22,50±15,41%, 56,33±8,98%, dan 42,50±6,35%; hasil MPU sebesar: 31,42±17,90%, 61,50±10,82%, dan 42,50±12,14%. Berdasarkan hasil penelitian dapat disimpulkan bahwa penambahan gliserol dalam pengencer Tris Kuning Telur dapat mempertahankan kualitas spermatozoa (motilitas, spermatozoa hidup, dan MPU) sapi aceh setelah pembekuan. Penambahan gliserol dengan konsentrasi 5% dalam pengencer Tris Kuning Telur paling baik dalam mempertahankan kualitas spermatozoa sapi aceh dibandingkan dengan konsentrasi 3% dan 7% (Freezing spermatozoa are often experiencing the damage by the formation of ice crystal. The formation of ice crystal leads to the quality reduction of post-thawing spermatozoa especially reducing motility. The addition of cryoprotectant in the diluent can minimize cell damage. One of the most common cryoprotectant type that was used in sement freezing is glycerol. This research aims to examine the influence of the addition of glycerol in tris-yolk, or known as Tris Egg Yolk (TKT) to the quality of spermatozoa (motility, live spermatozoa, and MPU) of aceh cattles stored in liquid nitrogen. The research design used in this research is a completely randomized design, that consists of three treatments and six repetitions. Quality of spermatozoa observation were performed after freezing process in the liquid nitrogen. Data were analyzed by Analysis of Variance (ANOVA) with one tail pattern. The result of spermatozoa quality in each concentration of glycerol (3%, 5%, and 7%) respectively for the motitity as follows: 17.17±12.17%; 51.17±11.13%; and 33.33±8.76%; result of live spermatozoa as follows: 22.50±15.41%; 56.33±8.98%; and 42.50±6.35%; result of MPU as follows: 31.42±17.90%; 61.50±10.82%; and 42.50±12.14%. Based on the result, the conclusion is that the addition of glycerol in the tris-yolk can maintain the quality of spermatozoa (motility, live spermatozoa, and MPU) of aceh cattle after freezing. The addition of glycerol with 5% concentration in the tris-yolk dilator is the best concentration to maintain the quality of spermatozoa aceh cattle, compared to the 3% and 7% concentration)