Aim: To evaluate the effects of melatonin supplementation of culture medium on the sperm survival rate, fertilization rate, cleavage rate, optimal embryo rate, blastocyst rate, implantation rate and clinical pregnancy rate. Material and Methods: Oocytes and sperm obtained from each patient (86 patients in total) were divided on average into four groups with different concentrations of melatonin (0 nmol/L, 0.1 nmol/L, 1.0 nmol/L, and 10.0 nmol/L). Melatonin was supplemented three times a day to maintain the concentration in different groups, and all the results were recorded at each related time. Results: The results indicated that melatonin could increase sperm survival rate, fertilization rate, and cleavage rate, and the solutions with 10 nmol/L melatonin were most effective (p < 0.05). Lower concentration of melatonin (0.1 nmol/L) might have negative effect on the fertilization rate. Melatonin could improve significantly optimal embryo rate, blastocyst rate, implantation rate, and clinical pregnancy rate (p < 0.05) and the peak values all appeared in the treatment group with 1.0 nmol/L melatonin. Conclusions: Addition of 1.0 nmol/L melatonin into culture media was the most effective to improve embryo quality and clinical outcome of IVF.