Like many steroid receptors, the glucocorticoid (GC) receptor (GR) is a phosphoprotein. Although there are multiple phosphorylation sites critical for GR transcriptional activity (i.e., serine [S]203, S211, and S226), their respective role in driving GR functions is highly cell specific. We have recently identified protein phosphatase 5 as an essential Ser/Thr phosphatase responsible for impairing GR function via S211 dephosphorylation in airway smooth muscle (ASM) cells. Because p38 mitogen-activated protein kinase (MAPK) directly phosphorylates GR in different cell types in a stimulus- and cell-dependent manner, we investigated the role of p38 MAPK on GR phosphorylation and function in ASM cells. Cells were transfected with 100 nM p38 MAPK small interfering RNA or 2 μg MAPK kinase 3 expression vector (a specific kinase that directly activates p38 MAPK) in the presence or absence of fluticasone (100 nM) and/or p38 MAPK pharmacological inhibitor SB203580. We found that p38 MAPK blockade positively regulates GR nuclear translocation and GR-dependent induction of the steroid-target gene GC-induced leucine zipper in a hormone-independent manner. We also found that p38 MAPK-dependent regulation of GR functions was associated with a differential action on GR phosphorylation at S203 and S211 residues. This study demonstrated that the inactive state of GR in resting conditions is not only ensured by the absence of the GC ligand but also by p38 MAPK-dependent phosphorylation of unliganded GR at specific residues, which appears to be important in determining the overall GC responsiveness of ASM cells.