The important role for the human small intestinal microbiota in health and disease has been widely acknowledged. However, the difficulties encountered in accessing the small intestine in a non-invasive way in healthy subjects have limited the possibilities to study its microbiota. In this study, a dynamic in vitro model that simulates the human ileum was developed, including its microbiota. Ileostomy effluent and fecal inocula were employed to cultivate microbial communities within the in vitro model. Microbial stability was repetitively achieved after 10 days of model operation with bacterial concentrations reaching on average 107 to 108 16S rRNA copy numbers/ml. High diversities similar to those observed in in vivo ileum samples were achieved at steady state using both fecal and ileostomy effluent inocula. Functional stability based on Short Chain Fatty Acid concentrations was reached after 10 days of operation using fecal inocula, but was not reached with ileostomy effluent as inoculum. Principal Components and cluster analysis of the phylogenetic profiles revealed that in vitro samples at steady state clustered closest to two samples obtained from the terminal ileum of healthy individuals, independent of the inoculum used, demonstrating that the in vitro microbiota at steady state resembles that of the human ileum.