Glucocorticoids are potent anti-inflammatory agents, acting through the glucocorticoid receptor (GR) to regulate target gene transcription. However, GR may also exert acute effects, including activation of signaling kinases such as c-Src and Akt, possibly via the scaffold protein, MNAR. MNAR inhibited GR transactivation in A549, but in HEK293 cells there was a ligand concentration dependent biphasic effect. Transactivation driven by low ligand concentrations was inhibited by MNAR expression whereas higher ligand concentrations were potentiating. Further analysis revealed that MNAR inhibited transactivation by the ligand independent AF1, but potentiated the C-terminal AF2 domain. The effect of MNAR was independent of c-Src activity, demonstrated by inhibitors and c-Src knockdown studies. In support of the role of MNAR in modulating GR transactivation, co-immunoprecipitation studies showed interaction between MNAR and GR in the nucleus but not the cytoplasm. Furthermore, MNAR and c-Src were also found to physically interact in the nucleus. Immunofluorescence studies showed MNAR to be predominantly a nuclear protein, with significant co-localization with GR. Deletion studies revealed that MNAR 874-1130 was co-immunoprecipitated with GR, and furthermore this fragment inhibited GR transactivation function when overexpressed. In addition, MNAR 1-400, which contains multiple LxxLL motifs, also inhibited GR transactivation. Taken together, MNAR interacts with GR in the nucleus but not cytoplasm, and regulates GR transactivation in a complex manner depending on cell type. MNAR is capable of regulating both AF1 and AF2 functions of the GR independently. MNAR expression is likely to mediate important cell variation in glucocorticoid responsiveness, in a c-Src independent mechanism.