The aim of this study was to investigate the effect of addition of cysteamine to the semen extender on post-thaw semen quality. A total of 30 ejaculates were collected from six bulls. Each ejaculate was divided into five equal parts and diluted to final concentration of 80 million sperms/mL using Optixcell (R) (IMV, France) semen extender supplemented with different concentrations of cysteamine (0, 0.75,1.25, 2.5 and 5 mM) and cryopreserved. In the frozen-thawed samples, the VAP, VSL, VCL ALH and sperm motility of control samples was greater (P<0.05) than cysteamine treated samples. The sperm abnormality and malondialdehyde (MDA) concentration were found highest in 5 mM cysteamine treated samples. The cysteamine treated samples travelled significantly less distance in cervical mucus as compared to control. Further, cysteamine decreased acrosomal integrity of sperm. In incubation test, control samples showed better sperm motility as compared to treatment groups. Further, cysteamine supplementation decreased the total antioxidants and increased the MDA concentration of sperm. From the study, we hypothesized that cysteamine cannot stimulate synthesis of glutathione (GSH) intracellularly in sperm to combat free radicals because during the maturation, sperm lost its cytoplasm which is necessary for biochemical reaction in which cysteamine reacts with cystine to form a mixed disulfide which taken up by cells and split into cysteine in the cytoplasm. Synthesis of GSH depends on the availability of cysteine. In conclusion, the results of our study strongly emphasize that cysteamine would not be a suitable additive in extender for freezing buffalo bull semen. (C) 2016 Elsevier B.V. All rights reserved.