The aim of this study was to evaluate the effect of estradiol (E2), oxytocin (OT), relaxin (relaximol) injection and prostaglandin E1 (misoprostol) suppository on the cervix dilation, time of cervical dilation persistency and pregnancy rate of Zandi ewes using artificial insemination. Eighty 3-4 years old and 55±2.5 kg weight ewes were used for this study. In experiment 1, the ewes were assigned to four equally groups. The groups were received 100 µl E2, 100 IU of OT, 5 ml relaximol and 200 µg misoprostol, respectively. Then, cervical dilation and time of cervical dilation persistency were examined at four times. E2 had no effect on cervical dilation and time of cervical dilation persistency (P>0.05). Using of OT, relaximol and misoprostol lead to cervical dilation (P<0.05). In experiment 2, the estrous synchronization was accomplished using CIDR insertion for 12 days. At the time of CIDR removal, 500 IU of eCG injected in to the ewes and then assigned to four equally groups. The control group received no cervical dilator treatment. Other three groups received the same treatment as the first experiment. According to the best time of cervical dilation in experiment 1, ewes were inseminated transcervically in the times of 20 and 40 min and 5 hours after treating, respectively, at 54 hours after eCG injection. Fifty days after insemination, pregnancy rate were recognized via ultrasonography diagnosis. Pregnancy rate in OT group was higher (P<0.05) than misoprostol and relaximol groups (60% vs 25 and 10%, respectively). However, there were no significant differences (P>0.05) between oxytocin and control group (60% vs 50%) in pregnancy rate. In conclusion, injection of oxytocin dilate the cervical canal and improved pregnancy rate in Zandi ewes, therefore this could be used as an instrument for improving pregnancy rate during artificial insemination.