Results: In order to determine the potential substrate preference of bifidobacteria, we compared the glycoside hydrolase (GH) profiles of a pooled-bifidobacterial genome (PBG) with a representative microbiome (RM) of the human gut. In bifidobacterial genomes, only 15% of GHs contained signal peptides, suggesting their weakness in utilization of complex carbohydrate, such as plant cell wall polysaccharides. However, compared with other intestinal bacteria, bifidobacteiral genomes encoded more GH genes for degrading starch and starch hydrolysates, indicating that they have genetic advantages in utilizing these substrates. Bifidobacterium longum subsp. longum BBMN68 isolated from centenarian’s faeces was used as a model strain to further investigate the carbohydrate utilization. The pathway for degrading starch and starch hydrolysates was the only complete pathway for complex carbohydrates in human gut. It is noteworthy that all of the GH genes for degrading starch and starch hydrolysates in the BBMN68 genome were conserved in all studied bifidobacterial strains. The in silico analyses of BBMN68 were further confirmed by growth experiments, proteomic and real-time quantitative PCR (RT-PCR) analyses.