A validated in vitro model of the large intestine (TIM-2), set up with human or pig feces, was used to evaluate the impact of potentially probiotic Lactobacillus amylovorus DSM 16698, administered alone (i), in the presence of prebiotic galactooligosaccharides (GOS) (ii), and both co-administered with probiotic Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12) (iii) on GOS degradation, microbial growth (L. amylovorus, lactobacilli, bifidobacteria and total bacteria) and metabolite production. High Performance Anion Exchange Chromatography revealed that GOS degradation was more pronounced in TIM-2 inoculated with pig feces compared to human feces. DGGE profiling of PCR-amplified 16S rRNA genes detected a more complex Lactobacillus spp. community in pig fecal material compared to human fecal inoculum. According to 16S rRNA gene-targeted qPCR, GOS stimulated the growth of lactobacilli and bifidobacteria in fecal material from both origins. The cumulative production of short chain fatty acids and ammonia was higher (P < 0.05) for pig compared to human feces. However, lactate accumulation was higher (P < 0.05) in the human model and increased after co-administration with GOS and Bb-12. This study reinforced the notion that differences in microbiota composition between target host organisms need to be considered when animal data are extrapolated to human, as it is often done with pre- and probiotic intervention studies © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.