Background: Pretreatment of the semen samples of the asthenozoospermia patients with certain sperm stimulant agents prior to use for assisted reproductive techniques (ART) has been reported to have beneficial effects on fertilization rates. Objectives: Investigating the influence of addition of different concentrations of oxytocin (OT) to semen samples of asthenozoospermic patients for in vitro sperm activation as a new method for stimulation of sperm motility in these patients and exploring the concentration of OT that harvests amaximal numberofmotile sperms. Methods: Simple layer technique for in vitro sperm activation using culture medium enriched with OT was studied in asthenozoospermic patients (No. 90). Semen samples were examined before sperm activation, and then they were divided into three equal groups. In the first group, three concentrations of OT (2, 4, and 6 IU/ml) were applied. Based on the results of the mentioned work, OTat the concentration of 2 IU/mlwas applied with sperm preparation medium (SPM) and without in the second and third groups, respectively. Results: Results showed that there was a significantimprovementin the percentageof sperm motilityand spermgrade activity of the OT-treated samples atthe concentration of 2IU/mlas comparedto the control samples. Moreover, there was a tendency for a decrease in sperm grade activity with increase OT concentration. The outcome of sperm activation in presence of SPM revealed a significant increase in the sperm motility characteristics (the percentage of sperm motility and sperm grade activity) in the control and OT-treated samples and the best improvement in these sperm motility characteristics was observed in the OT-treated samples. When SPM was absent, a significant improvement in the sperm motility characteristics was observed only in the OTtreated samples. Conclusion: The results of this study indicate that OT at the concentration of 2 IU/ml has got a stimulatory effecton sperm motility characteristics; also, the use of SPM succeeds to supportthis effect.