Mizoribine (MIZ) is a novel imidazole nucleoside with immunosuppressive activity. MIZ has been approved in japan and combination therapy with MIZ and glucocorticoids has been used after renal transplantation and for lupus nephritis and rheumatoid arthritis. In this study, we identify 14-3-3 proteins as MIZ-binding proteins. 14-3-3 proteins interact with many proteins involved in cellular signaling, including the glucocorticoid receptor (GR). The 14-3-3/GR interaction enhances the transcriptional activity of the receptor. We show that MIZ affects the conformation of 14-3-3 proteins and enhances the interaction of GR and 14-3-3 eta dose dependently in vitro. MIZ also has a stimulatory effect on transcriptional activation by the GR. Our results point to the possibility that one mechanism for the therapeutic effect of MIZ could he to regulate the GB function via 14-3-3 proteins.