Faecalibacterium prausnitzii, an abundant member of the human commensal microbiota, has been proposed to have a protective role in the intestine. However, it is an obligate anaerobe, difficult to co-culture in viable form with oxygen-requiring intestinal cells. To overcome this limitation, a unique apical anaerobic model of the intestinal barrier, which enabled co-culture of live obligate anaerobes with the human intestinal cell line Caco-2, was developed. Caco-2 cells remained viable and maintained an intact barrier for at least 12¿h, consistent with gene expression data, which suggested Caco-2 cells had adapted to survive in an oxygen-reduced atmosphere. Live F.¿prausnitzii cells, but not ultraviolet (UV)-killed F.¿prausnitzii, increased the permeability of mannitol across the epithelial barrier. Gene expression analysis showed inflammatory mediators to be expressed at lower amounts in Caco-2 cells exposed to live F.¿prausnitzii than UV-killed F.¿prausnitzii, This, consistent with previous reports, implies that live F.¿prausnitzii produces an anti-inflammatory compound in the culture supernatant, demonstrating the value of a physiologically relevant co-culture system that allows obligate anaerobic bacteria to remain viable.