Clinical and experimental observations have long suggested that elevated levels of estrogen associate with increased serum iron availability. Additionally, recent work has shown that estrogen can downregulate hepcidin synthesis in vitro. This study aims at assessing whether the ability of estrogen to downregulate hepcidin synthesis translates into changes in serum iron status. Hepcidin synthesis was evaluated in MCF-7, Hep-G2 and SKOV-3 cells treated with increasing concentrations of estrogen and cultured for up to 24 h post treatment. The correlation between levels of serum estrogen, hepcidin and iron was assessed using serum samples collected from 153 premenopausal women at random and samples collected from 6 women at days 1, 5, 10, 16, 21 and 28 of the monthly cycle. Estrogen-treated MCF-7 cells showed a significant reduction in hepcidin synthesis, especially at 20 nM/24 h E2 treatment. Hepcidin synthesis was also significantly reduced in Hep-G2 and SKOV-3 cells at 20 nM/24 h E2 treatment. In serum samples collected at random, estrogen (P=0.022; R=-0.213) and iron (P=0.028; R=-0.316) correlated negatively with hepcidin and positively with each other (P=0.033; R=0.319). An overall similar pattern was also observed in monthly cycle-timed samples. These findings suggest that elevated levels of estrogen reduce hepcidin synthesis as means of enhancing serum iron content in menstruating women.