The objective of this study was to evaluate the characteristics of Korean Native Cattle sperm frozen-thawed with L-cysteine and/or catalase. The semen from bulls was collected by the artificial vagina method, and Triladyl containing 20% egg-yolk and/or L-cysteine (L), catalase (C) and L-cysteine + catalase was added to the diluted semen for cryopreservation. The results showed that sperm viability was significantly higher in the L-cysteine + catalase (69.49 ± 3.16%) group than in the control (60.5 ± 3.94%) group (p<0.05). Acrosome damage was significantly lower in the L-cysteine (17.12 ± 1.08%) group than in the control (21.46 ± 1.14%), catalase (20.54 ± 0.76%), and L-cysteine + catalase (19.29 ± 0.65%) groups (p<0.05). In addition, the level of intact mitochondria in the spermatozoa was significantly higher in the L-cysteine (58.65 ± 1.39%) group than in the control (50.63 ± 2.37%) group (p<0.05). The hydrogen peroxide level in the frozen-thawed sperm was significantly lower in the L-cysteine (3.74 ± 1.66%), catalase (4.65 ± 1.87%), and L-cysteine + catalase (8.11 ± 2.15%) groups than in the control (13.22 ± 1.6%) group (p<0.05). The glutathione level was significantly higher in the L-cysteine (1.33 ± 0.03%) group than in the control (1.08 ± 0.06 %), catalase (1.05 ± 0.02%) and L-cysteine + catalase (1.11 ± 0.03%) groups (p<0.05). In conclusion, L-cysteine and catalase could protect the membrane of Korean Native Cattle sperm from damage during sperm cryopreservation. Especially, L-cysteine was more effective for keeping acrosomes and mitochondria intactness during sperm cryopreservation.