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A validated in vitro gut model (i.e. SHIME®) was used to assess the effect of repeated daily administration of lactulose on microbial metabolic activity and community composition in different colonic areas, with the focus on inter-individual differences among three human subjects. An initial rise in acetate and lactate levels was observed in the proximal colon after lactulose administration, which could be linked to an overall strong bifidogenic effect as well as higher Lactobacilli levels in donors 2 and 3. Particularly two operational taxonomic units (OTUs) related to Bifidobacterium adolescentis and Bifidobacterium longum increased with lactulose addition. The enhanced acetate and lactate production subsequently stimulated microbial species involved in cross-feeding interactions, resulting in the donor-dependent production of propionate and/or butyrate. Additionally, a reduction in markers of proteolytic fermentation was detected upon lactulose supplementation. A wide spectrum of propionate- and especially butyrate-producing microbes, such as the next-generation probiotics Faecalibacterium prausnitzii and Akkermansia muciniphila, were donor-dependently enhanced in the distal colon, which is of specific interest as many colonic diseases originate in the distal part of the colon. For the first time beneficial effects of lactulose on the microbiota as well as metabolic activity could be demonstrated over the entire colon in vitro.