Background: Empathy is an important factor in patient-physician relationship that has beneficial effects in medical practice. Jefferson Scale of Empathy (JSE) has been specifically designed to assess empathy in health professionals (HP-version) and related students (S-version). Few validation studies have been performed on S-version of the scale. Aims: To examine empathy in a large sample of Iranian medical students, and also to study factor structure and psychometric properties of the Persian translation of the S-version of the JSE. Method: 1187 medical students (759 female) from 16 universities around the country participated in the study during 2009. Independent sample t-test, multivariate analysis of variance, and confirmatory factor analysis (CFA) were performed to analyze the data. Results: Female students outscored male students in empathy. The trend for empathy had a negative relationship with years of education. JSE showed an acceptable internal consistency and test re-test reliability. CFA upheld the original three-factor structure -Perspective Taking, Compassionate Care, and Standing in the Patient's Shoes -consisting 20 items. Conclusion: The decrease in empathy during medical education is consistent with previous studies. The Persian version of JSE is a valid and reliable measure to tap empathy in a Persian-speaking medical student.