The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N-terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chleator of intracellular divalent cations, and curcumin, a natural product with anti-inflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (DRIP150). Interestingly, in U2OS cells expressing a non-phosphorylated GR mutant S211A, the expression of IGFBP1 and IRF8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild type receptor across a broad range of hormone concentrations. In contrast, the induction of GILZ, a MED14-independent GR target, was similar in S211A- and wt GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and in some cases dependent upon the amount of activated receptor.