Intestinal epithelial cell (IEC) activation by non-pathogenic, commensal bacteria was demonstrated to require the presence of immunocompetent cells. In this study, HT-29 and CaCO-2 transwell cultures, reconstituted with CD4+ and CD8+ T cells, CD19+ B cells and CD14high monocytes, were challenged with non-pathogenic Gram negative Escherichia coli and Gram positive lactobacilli. Cytokine expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assays (ELISA). Expression of tumour necrosis factor alpha (TNF-alpha) and interleukin (IL)-8 mRNA in E. coli or L. sakei challenged IEC was promoted by lymphocyte populations predominantly CD4+ T cells, while monocytes failed to mediate an inflammatory cytokine response. The monocyte phenotype and function were further characterised by flow cytometry and mixed lymphocyte reaction (MLR). During the co-culture with IEC and bacterial stimulated IEC, CD14high peripheral blood monocytes acquired a CD14low CD16low phenotype with reduced expression co-stimulatory (CD80, CD86, CD58) cell surface molecules. Immunosuppressive functions of IEC conditioned CD14low monocytes were demonstrated by the predominant secretion of IL-10 and IL-IRa and their reduced potential to trigger an allogeneic lymphocyte response. In conclusion, IEC contribute to the development of CD14low CD16low monocytes with immunosuppressive function and antagonised a lymphocyte-mediated activation of the intestinal epithelium in response to intestinal and food derived bacteria. These results strengthen the hypothesis that the gut epithelium constitutes an important functional element in the regulation of mucosal immune homeostasis towards commensal bacteria.