We have investigated the effects of anthrax lethal toxin on human epidermal keratinocytes in the presence of factors associated with infection by gram-positive bacteria. Similar to previous results reported with alveolar macrophages, lethal toxin cleaved members of the mitogen activated kinase kinase (MEK) family of proteins, including MEK2 and MEK4, but not MEK5, in keratinocytes. Cleavage of MEK proteins was accompanied by inhibition of cell growth in keratinocyte cultures, but no biomarkers of apparent apoptosis or necrosis could be detected. When keratinocytes were incubated with lethal toxin alone, increases in secreted levels of both RANTES and MCP-1 could be detected by protein microarray analysis of cellular supernatants. When keratinocytes were incubated in the presence of a mixture of the pro-inflammatory mediator interferon gamma (IFNγ) and the NOD2 ligand, muramyl dipeptide (MDP), a broad range of inflammatory biomarkers were released into the surrounding medium. Addition of lethal toxin in the presence of IFNγ and MDP resulted in attenuation of the levels of a number of these biomarkers, e.g. IL-6, TNF-α, GM-CSF and MIP-1β, released into the medium. Analysis of transcription factors known to be activated at least in part via the mitogen activated protein kinase (MAPK) pathway indicated that intracellular levels of activated forms of c-Myc and c-Jun were decreased while levels of activated Stat-1α and the p65 subunit of activated members of the NF-κB family were increased after incubation of keratinocytes with lethal toxin. These data reveal the complexity of the response of keratinocytes to pro-inflammatory signals associated with infection by Bacillus anthracis and the modulation of that response by the actions of lethal toxin.