Impaired circulating estrogen levels have been related to impaired glycemic homeostasis and diabetes mellitus (DM), both in females and males. However, for the last twenty years, the relationship between estrogen, glycemic homeostasis and the mechanisms involved has remained unclear. The characterization of estrogen receptors 1 and 2 (ESR1 and ESR2) and of insulin-sensitive glucose transporter type 4 (GLUT4) finally offered a great opportunity to shed some light on estrogen regulation of glycemic homeostasis. In this manuscript, we review the relationship between estrogen and DM, focusing on glycemic homeostasis, estrogen, ESR1/ESR2 and GLUT4. We review glycemic homeostasis and GLUT4 expression (muscle and adipose tissues) in Esr1−/− and Esr2−/− transgenic mice. We specifically address estradiol-induced and ESR1/ESR2-mediated regulation of the solute carrier family 2 member 4 (Slc2a4) gene, examining ESR1/ESR2-mediated genomic mechanisms that regulate Slc2a4 transcription, especially those occurring in cooperation with other transcription factors. In addition, we address the estradiol-induced translocation of ESR1 and GLUT4 to the plasma membrane. Studies make it clear that ESR1-mediated effects are beneficial, whereas ESR2-mediated effects are detrimental to glycemic homeostasis. Thus, imbalance of the ESR1/ESR2 ratio may have important consequences in metabolism, highlighting that ESR2 hyperactivity assumes a diabetogenic role.