A glucocorticoid receptor (GR) interacting protein, interferon-inducible protein P56, was isolated from the human bone marrow cDNA library by two-hybrid screening in yeast using the GR ligand-binding domain (GR-LBD) as bait. The interaction between GR and P56 and the effect of P56 on GR were investigated. PCR was performed to amplify GR-LBD fragments and it was cloned into the bait vector pGBKT7 to create the plasmid pGBKT7-GR LBD. The plasmid was used as bait to screen a cDNA library constructed in the pACT2 vector. The positive colonies were sequenced. P56 and GR-LBD cDNA fragments were cloned respectively into the vector pGEX-4T-2, pACT2,pCMV-Myc, pCMV-HA for GST pull down, yeast two-hybrid, coimmunoprecipitation analysis and CAT activity assay. 42 positive clones were obtained by yeast two-hybrid, in which one isolated cDNA from the library encoded the COOH-terminal portion of the interferon inducible protein P56 (221～1 642 bp, residues 53～478 amino acids) as shown by DNA sequencing. Yeast two-hybrid, GST pull down and CO-IP assays verified that P56 interacted with GR-LBD. Expression of P56 resulted in dose-dependent decrease in GR-CAT expression when GRα, GRE-driven reporter genes were cotransfected with P56. Thus, the study demonstrates that P56 interacts with GR-LBD in vitro and in vivo, P56 inhibites the GR-mediated transcriptional activity.