Objective: The present study aimed to determine the prevalence of anti-syphilis antibodies in Iranian pregnant women who attended antenatal care (ANC) in 21 districts affiliated to Isfahan University of Medical Sciences (MUI) from April 2017 to April 2020. Materials and methods: In a cross-sectional study in 2020, the data about anti syphilis antibody were collected from electronic health files of pregnant women. Pregnancy specific health package was delivered to Iranian pregnant women in a horizontally integrated network across the country. The electronic health file was used for every Iranian citizen and all health events were recorded by their health care providers in 2016. All health services, particularly laboratory tests that were performed either in public or private laboratories, that were given to pregnant women needed to be recorded in their electronic health files. According to the pregnancy health package, they were screened initially by Rapid Plasma Reagin (RPR) test. Results: In those years, 70512, 72298, 63272 pregnant women were screened respectively. In these intervals, using the RPR test, no positive cases of anti- syphilis antibody were found. Conclusion: It seems that syphilis infection among Iranian pregnant women who attended ANC in districts affiliated to MUI has reached the elimination phase.