Animal studies have shown that endogenous estrogens such as 17β-estradiol (E2) can modulate lipid profiles in vivo, and this effect is generally thought to be mediated by the estrogen receptors (ERs). The present study sought to test a hypothesis that some of the endogenous estrogen metabolites that have very weak estrogenic activity may exert some of their modulating effects on lipid metabolism in an ER-independent manner. Using ovariectomized female rats as an in vivo model, we found that 4-hydroxyestradiol (4-OH-E2) has a markedly stronger effect in reducing the adipocyte size and serum cholesterol level in rats compared to E2, despite the weaker estrogenic activity of 4-OH-E2. Moreover, when E2 or 4-OH-E2 is used in combination with ICI-182,780 (an ER antagonist), some of their lipid-modulating effects are not blocked by this antiestrogen. Interestingly, two of the O-methylation metabolites of 4-OH-E2, namely, 4-methoxyestradiol and 4-methoxyestrone, which have much weaker estrogenic activity, were also found to have similar lipid-modulating effects compared to 4-OH-E2. Mechanistically, up-regulation of the expression of leptin, cytochrome P450 7A1 and LXRα genes is observed in the liver of animals treated with E2 or 4-OH-E2, and the up-regulation is essentially not inhibited by co-treatment with ICI-182,780. These results demonstrate that some of the endogenous E2 metabolites are functionally important modulators of lipid metabolic profiles in vivo. In addition, our findings indicate that an ER-independent pathway likely mediates some of the lipid-modulating effects of endogenous estrogens and their metabolic derivatives.