Mycoplasma fermentans (M. fermentans) was shown to be involved in the alteration of several eukaryotic cell functions (i.e. cytokine production, gene expression), and was suggested as a causative agent in arthritic diseases involving impaired apoptosis. We investigated whether M. fermentans has a pathogenic potential by affecting tumor necrosis factor (TNF)alpha-induced apoptosis in the human myelomonocytic U937 cell line. A significant reduction in the TNFalpha-induced apoptosis (approximately 60%) was demonstrated upon either infection with live M. fermentans or by stimulation with non-live M. fermentans. To investigate the mechanism of M. fermentans antiapoptotic effect, the reduction of mitochondrial transmembrane potential (DeltaPsim) and the protease activity of caspase-8 were measured. In the infected cells, the reduction of DeltaPsim was inhibited (approximately 75%), and an approximately 60% reduction of caspase-8 activity was measured. In conclusion, M. fermentans significantly inhibits TNFalpha-induced apoptosis in U937 cells, and its effect is upstream of the mitochondria and upstream of caspase-8.