Background: There are no randomized controlled trials evaluating the pregnancy rates after thawed blastocyst transfers in patients treated with various hormone replacement regimens. Methods: A prospective randomized controlled trial was conducted to evaluate the outcomes in three different hormone replacement protocols for thawed blastocyst transfer. A total of 330 women (median age 38.2 years) who were undergoing IVF at our clinic were enrolled. Results: Serum estradiol (E2) levels were 267.71 pg/ml in Premarin group, 391.22 pg/ml in Estrogel group and 495.12 pg/ml in Estrana tape group. Therefore, serum E2 levels in Estrana tape group were higher than those of the other two groups (P<0.01). The pregnancy rate in the Estrogel group was higher than that in the Premarin group (30.0% versus 17.3%, P=0.026, odds ratio 2.05, 95% confidence interval: 1.09-3.87). Furthermore, the pregnancy rate in the Estrana tape group was higher than that in the Estrogel group (43.6% versus 30.0%, P=0.036, odds ratio 1.81, 95% confidence interval: 1.04-3.14). Conclusion: The serum E2 levels contributed to differences observed in the pregnancy rate among the three different protocols. Thus, Estrana tape has an advantage as a hormone replacement protocol for thawed blastocyst transfer. Clinical trial registration ID #UMIN000016919.