MKP-M is a dual specificity phosphatase that preferentially inactivates JNK. mkp-M gene expression is rapidly induced by lipopolysaccharide (LPS) stimulation in macrophages and is involved in the negative regulation of LPS-mediated JNK activation and tumor necrosis factor-alpha secretion. To reveal the transcriptional regulation of the mkp-M gene, we isolated the mouse mkp-M gene and mapped its transcriptional start site. Luciferase reporter plasmids containing 5'-upstream regions of the mkp-M gene were stably transfected into RAW264.7 cells. The assays using these cells revealed that the promoter region between -252 and -135 is required for mkp-M promoter activation. Sequencing analysis revealed E box and CREB-responsive elements in this region, and electromobility shift assays and mutagenesis confirmed that both of these elements are essential for LPS responsiveness of the mkp-M gene. We also utilized chromatin immunoprecipitation assay and found that LPS stimulation caused acetylation of histone H3 and H4 at mkp-M promoter in RAW264.7 cells. Consistent with this, a histone deacetylase inhibitor, trichostatin A, increased endogenous mkp-M gene transcription. Finally, DNase I hypersensitivity site mapping revealed the inducible hypersensitivity site after LPS stimulation around the location of the E box and CREB-responsive elements. Altogether, our data indicated that the activation of mkp-M gene transcription in macrophages by LPS is associated with histone acetylation and chromatin remodeling.