Our previous studies demonstrated that premenopausal women have dominant vagal and subordinate sympathetic activity compared with age-matched men. This study was designed to investigate the role of estrogen in gender-related autonomic differences. We evaluated the heart rate variability of four healthy groups: age-matched postmenopausal women without hormone replacement therapy (PM), postmenopausal women on conjugated estrogen replacement therapy (PME), men, and non-age-matched premenopausal women (PreM). Frequency-domain analysis of short-term and stationary R-R intervals was performed to evaluate low-frequency power (LF; 0.04-0.15 Hz), high-frequency power (HF; 0.15-0.40 Hz), the ratio of LF to HF (LF/HF), and LF in normalized units (LF%). No gender-related autonomic differences existed between the PM and men groups, but they did exist between the PME and men group. Compared with the PreM group, the PM group had a lower HF and higher LF% and LF/HF. Compared with the PM group, the PME group had a higher HF but lower LF% and LF/HF. These results suggest that conjugated estrogen replacement therapy may facilitate vagal and attenuate sympathetic regulation of heart rate in postmenopausal women. In addition, estrogen may play an important role in gender-related autonomic differences.