The effects of Lactobacillus acidophilus NCFM™, lactitol, and the combination of lactitol and L. acidophilus NCFM™ were studied with a semi-continuous colon fermentation simulation; consisting of compartments mimicking, ascending, transverse, descending and sigmoid colon and their conditions with faecal inoculation. L. acidophilus NCFM™ was detected throughout the colon simulator. Lactitol was utilised early on by the microbes in the proximal part of the simulator. Lactitol increased the total numbers of microbes and bifidobacteria, and decreased clostridia cluster IV, while L. acidophilus NCFM™ alone decreased the numbers of clostridia cluster XIV. Combination treatment increased the numbers of bifidobacteria. Furthermore, concentrations of acetic acid, butyric acid and the sum of total short-chain fatty acids were increased by both lactitol-including treatments. The treatment with L. acidophilus NCFM™ alone increased the concentration of propionic acid and butyric acid. L. acidophilus NCFM™ tended to increase the total concentrations of biogenic amines, while lactitol suppressed production of biogenic amines also in the presence of L. acidophilus NCFM™. True synergistic effects are suggested in stimulation of the production of butyrate, an important microbial metabolite for colon health. In conclusion, lactitol as well as the combination of lactitol and L. acidophilus NCFM™ were found to exhibit complementary beneficial effects on the colon microbial composition and activity.