The production of reactive oxygen species (ROS) during cryopreservation of semen alters the sperm motion and mobility characteristics, resulting in poor or failure of conception rate after artificial insemination (AI). Melatonin an antioxidant is able to modulate the effect of ROS and prevents spermatozoa by reducing the oxidative stress during freezing process. Eight ejaculates from eight healthy HF bulls diluted with Tris egg yolk glycerol extender were divided into five equal aliquots. The Computer Assisted Semen Analyser (CASA) results showed no significant difference between the control—post‐ thaw samples and melatonin‐treated samples; however, the velocity of spermatozoa with regard to the VAP, VSL showed highest increase in the 0.25 mM MLT‐treated semen followed by 0.1 mM MLT treated semen except for VCL where velocity increased with increase in the concentration of melatonin. The vigour of spermatozoa regard to BCF, STR and LIN recorded highest increase in the 0.25 mM MLT treated semen followed by 0.1 mM MLT‐treated semen except for the ALH where vigour increased with increase in the concentration of melatonin. The electron micrography images illustrated that the addition of 0.1 mM melatonin protected the plasma membrane and acrosome region and maintained the ultrastructure integrity of the cryopreserved spermatozoa when compared to control group, whereas the electron micrography of spermatozoa treated with 0.2 and 0.25 mM melatonin illustrated highest damage to the plasma and acrosome membrane. Thus concluding that inclusion of melatonin to sperm extender can improve the post‐thaw quality of cryopreserved bull spermatozoa.