Purpose: To evaluate the effects of estradiol (E2) supplementation on pregnancy outcome in patients with unexplained infertility undergoing in vitro fertilization (IVF). Materials and Methods: A total of 100 women with unexplained infertility and candidates for IVF, were included in this study and were randomly assigned to receive E2 supplementation or placebo during the luteal phase. The E2 serum levels in the hCG administration day and third and seventh day after ovum retrieval were measured in control group. The rate of pregnancy was also quantified and compared between the two study groups. Results: There was no significant difference between two study groups regarding baseline characteristics. E2 level decreased significantly in third (1765.34 ± 680.09; p < 0.001) and seventh (1459.66 ± 593.80; p < 0.001) days after ovum retrieval (2411.16 ± 713.52). The authors found that the serum level of E2 was significantly lower in those who received E2 supplementation at day 3 (p < 0.001) and 7 (p<0.001). However the pregnancy rate was not significantly different between two study groups (p = 0.849). In the same way, there was no significant difference between two study groups regarding the number of retrieved oocytes (p = 0.563) and number of MII oocytes (p = 0.103). Conclusions: E2 supplementation during the luteal phase in patients with unexplained infertility undergoing IVF, is associated with decreased serum levels of E2 after hCG injection. However the fertility outcome was not affected by E2 supplementation.