We have recently identified the MAPK phosphatase (MKP)-1 as a novel mediator of the anti-inflammatory properties of glucocorticoids (dexamethasone) in the human endothelium. However, nothing is as yet known about the signaling pathways responsible for the upregulation of MKP-1 by dexamethasone in endothelial cells. Knowledge of the molecular basis of this new alternative way of glucocorticoid action could facilitate the identification of new anti-inflammatory drug targets. Thus, the aim of our study was to elucidate the underlying molecular mechanisms. Using Western blot analysis, we found that dexamethasone rapidly activates ERK, JNK, and p38 MAPK in human umbilical vein endothelial cells. By applying the kinase inhibitors PD98059 (MEK1) and SP600125 (JNK), ERK and JNK were shown to be crucial for the induction of MKP-1. Using electrophoretic mobility shift assay and a decoy oligonucleotide approach, the transcription factors AP-1 (activated by ERK and JNK) and CREB (activated by ERK) were found to be involved in the upregulation of MKP-1 by dexamethasone. Interestingly, dexamethasone induces the generation of reactive oxygen species (measured by dihydrofluorescein assay), which participate in the signaling process by triggering JNK activation. Our work elucidates a novel alternative mechanism for transducing anti-inflammatory effects of glucocorticoids in the human endothelium. Thus, our study adds valuable information to the efforts made to find new anti-inflammatory principles utilized by glucocorticoids. This might help to gain new therapeutic options in order limit glucocorticoid side effects and to overcome resistance.