To test the hypothesis that increased circulating estradiol increases hepatic glutamine synthetase (GS) content in young and aged cows, and two nonclassical nongenomic estradiol pathway intermediates [β-catenin, G protein-coupled receptor 30 (GPR30)], 12 young (3–4 yr) and 12 aged (≥8 yr) cows were randomly allotted (n = 6) and received either none or estradiol (TRT) implants (Compudose^®) for 28 d. Blood and liver samples were collected on days 16 and 28 and the effects of TRT, cow age (age), time after implant (day), and their interactions were analyzed by analysis of variance (ANOVA). Plasma estradiol was increased by TRT in young and aged cows on days 16 and 28 and on day 28 vs. day 16. An age × day interaction (P = 0.01) revealed lower GS mRNA at day 28 in young cows but increased mRNA in aged cows. Aged cows had less GS protein than young cows, and a TRT × day interaction (P = 0.02) reflected 134% more of GS on day 16 in TRT in aged cows. No main or interaction effects were found for progesterone, β-catenin, or GPR30. We conclude that hepatic GS expression is transiently stimulated by increased circulating estradiol, whereas expression of β-catenin or GPR30 was not affected.