The effects of melatonin upon the bull liquid semen and enzymatic release in seminal plasma during storage at 4°C were studied. The semen was extended by using Russian diluent to obtain 100 X 10° sperm per ml. The melatonin was added to extended semen as 0.0, 1.0, 5.0, 10.0, 15.0 and 20.0 ug /100 X 10° sperm. The samples in this study were stored at 4°C for six days and examined daily for motility, alive, abnormalities % as well as fructose content, Acid phosphatase, ATPase and hyaluronic acid in seminal plasma. The results in this study demonstrated that melatonin, particularly the high concentrations (10.0, 15.0 and 20.0 ug), improved sperm motility (P<0.01) for a long period of storage, reduced dead sperm % (P <0.01) as well as conservation of energy in seminal plasma (P<0.01). The ultrastructure investigation of the treated bull semen after 3 and 5 days storage revealed that, the high concentration (especially at 20 ug) melatonin had a good protective effect for sperm. The effect of melatonin on sperm cells may be through activation of microtubular function, protection of DNA from damage by free radical moreover, it was antiaging on sperm cells. These results also arise the possibility of using the melatonin with the extender or diluent (especially at 20ug / 100 X 10° sperm per ml) for improving the liquid semen quality and storage for long time at 4°C.