We investigated the mechanisms by which estrogen alters insulin signaling in 3T3-L1 adipocytes. Treatment with 17-estradiol (E2) did not affect insulin-induced tyrosine phosphorylation of insulin receptor. Estradiol enhanced insulin-induced tyrosine phosphorylation of IRS-1, IRS-1/p85 association, phosphorylation of Akt, and 2-deoxyglucose uptake at 10^-8 M but inhibited these effects at 10^-5 M. 10^-5 M estradiol enhanced insulin-induced phosphorylation of IRS-1 at Ser (307), which was abolished by treatment with a JNK inhibitor. In addition, the effect of estradiol was abrogated by pretreatment with a specific estrogen receptor antagonist, ICI182,780. Membrane-impermeable estradiol E2-BSA did not affect the insulin-induced phosphorylation of Akt at 10^-8 M but inhibited it at 10^-5 M. Furthermore, estradiol decreased the amount of estrogen receptor at the plasma membrane at 10^-8 M but increased it at 10^-5 M. In contrast, the subcellular distribution of estrogen receptor was not altered by the treatment. These results indicate that estradiol affects the metabolic action of insulin in a concentration-specific manner, that high concentrations of estradiol inhibit insulin signaling by modulating phosphorylation of IRS-1 at Ser (307) via a JNK-dependent pathway, and that the subcellular redistribution of estrogen receptor in response to estradiol may explain the dual effect of estradiol.