Prebiotics, such as short-chain fructo-oligosaccharides (scFOS), have been shown to modify colonic microflora composition and activity in many humans and some animal species. However, little data are available on their fermentation characteristics by the ileal microbial community, and their effects on the small intestinal microflora composition. The aim of this study was to evaluate the in vitro fermentability of scFOS by ileal bacteria and the effect of scFOS on concentrations of the total anaerobic bacteria, Lactobacilli, Streptococci, Bifidobacteria and Clostridium perfringens in veal calf ileal contents. Two groups of 4 calves fed a finisher milk replacer and fibrous pellets were sacrificed 5 h after the morning meal. In each group, ileal contents were pooled and diluted with Lowe's medium. Two amounts of scFOS (100 and 250 mg) were tested in vitro versus a control (0 mg scFOS). Each bottle was incubated in duplicate for each treatment and each time point at 37 °C in a shaking water bath. Bacterial concentrations were determined at 0, 3, 6, and 9 h and pH, gas production, volatile fatty acids (VFA), and lactate concentrations were measured every 1.5 h for 9 h. Over time, scFOS fermentation resulted in more gas production (P=0.055), higher total VFA concentrations (P<0.01), an increase in l-lactate concentration (P<0.01), as well as a lower pH value (P<0.01) than the control bottle. Acetate and butyrate concentrations increased (P<0.01) with scFOS fermentation. ScFOS fermentation enhanced growth of Lactobacilli (P=0.010), Streptococci (P=0.015) and lactic acid utilizing bacteria (P=0.025). C. perfringens concentration was not affected by scFOS addition. Based on our results, scFOS was fermented by bacteria of ileal contents in veal calves and its fermentation led to a selective stimulation of host bacteria. However, further studies are needed to determine impacts of scFOS on C. perfringens concentration in veal calf ileal contents.