Mycoplasma have been shown to be involved in the alteration of several eukaryotic cell functions, such as cytokine production, gene expression and more. We have previously reported that infection of human myelomonocytic U937 cell line with live Mycoplasma fermentans (M. fermentans) inhibited tumour necrosis factor (TNF-alpha)-induced apoptosis. Mycoplasmal membrane lipoproteins are considered to be the most potent initiators of inflammatory reactions in mycoplasmal infections. The aim of this study was to clarify whether the inhibitory effect on TNFα-induced apoptosis is exerted by M. fermentans lipoproteins (LPMf). A significant reduction in TNFα-induced apoptosis was demonstrated by stimulation of U937 cells with M. fermentans total proteins, LPMf or MALP-2 (M. fermentans synthetic lipopeptide), but not with M. fermentans hydrophilic protein preparation (AqMf). To investigate the mechanism of M. fermentans antiapoptotic effect, the reduction of mitochondrial transmembrane potential (Δψm) was measured. M. fermentans total proteins LPMf and MALP-2, but not AqMf, inhibited the reduction of Δψm. In addition, M. fermentans total proteins LPMf and MALP-2, but not AqMf, downregulated the formation of active caspase-8. NF-κB was transactivated in cells treated with M. fermentans lipoproteins, and was essential for host cell survival, but not for the inhibition of TNFα-induced apoptosis by LPMf. Our results suggest that the inhibitory effect exerted by M. fermentans on TNFα-induced apoptosis in U937 cells is due to the membrane lipoproteins of these bacteria.