It is established that increases in neuropeptide Y (NPY) expression are associated with hyperphagia and obesity. These effects can be reversed by estrogen, a recognized anorexigen. We found that 17β-estradiol (E₂) regulates biphasic NPY gene expression in a clonal, immortalized hypothalamic cell line, N-38, through estrogen receptor (ER) action at the level of the NPY promoter. However, rapid, nongenomic actions of estrogen, linked to the phosphatidylinositol 3-kinase (PI3-K)/Akt and ERK1/2 mitogen-activated protein kinase (MAPK) pathways, may also play a role. We therefore examined the changes in the phosphorylation status of Akt, ERK1/2, and cAMP response element-binding protein (CREB) after treatment with 10 nm E₂ in the N-38 neurons and found activation of these signaling proteins within 5–30 min. We also demonstrated possible cross talk between the estrogen-activated PI3-K/Akt and MAPK/extracellular signal-regulated kinase pathways using pharmacological inhibitors. We find that only ERα is involved in the early signaling events using the ERα agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and the ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile. Furthermore, we can detect colocalization of ERα and caveolin-1, a membrane-associated signaling protein. Remarkably, we find that the membrane-mediated events are critical for the long-term estrogen-mediated repression of NPY gene expression that can be mapped to within −97 bp of the NPY promoter. To link the early signaling events to downstream effectors, we detected induction of c-fos and inactivation of MSK-1 by estrogen and binding of CREB to this minimal promoter region. These observations suggest that rapid estrogen-mediated signaling is mediated by ERα, and the signal transduction events potentiate the genomic actions of estrogen on NPY gene expression in the N-38 NPY neurons.