The glucocorticoid receptor (GR), a member of the nuclear receptor superfamily, mediates the effects of glucocorticoids. It is known that 14-3-3 family proteins interact with GR and regulate its transcriptional activity. They also bind to several molecules and influence many cellular events by altering their subcellular localization and/or acting as a chaperone. Recently, it has been proposed that ligand-activated degradation of GR occurs via the ubiquitin-proteasomal degradation pathway and that inhibition of proteasomal activity induces up-regulation of GR and enhances the transcriptional activity of GR. To examine the function of 14-3-3eta in the glucocorticoid-dependent signal pathway, we studied the regulatory role of 14-3-3eta in ligand-induced GR transcriptional activation. 14-3-3eta Enhanced the transcriptional activity of GR, and the levels of GR were higher in cells transfected with the 14-3-3eta expression vector in response to glucocorticoid. The GR level increased in both cytosol and nucleus, and endogenous GR was also elevated by 14-3-3eta in HeLa cells. 14-3-3eta Inhibited ligand-induced down-regulation of GR. Proteasomal inhibition did not induce any synergistic effect on the 14-3-3eta-induced increase in GR in response to glucocorticoid, and inhibition of translation did not block elevation of GR by 14-3-3eta, indicating that 14-3-3eta induces stabilization of GR. These results suggest that 14-3-3eta functions as a positive regulator in the glucocorticoid signal pathway by blocking the degradation of GR and inducing an elevation of GR, thus enhancing the transcriptional activity of GR.