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Summary: Diet-microbe interactions play a crucial role in modulation of the early life microbiota and infant health. Bifidobacterium dominates the breast-fed infant gut and may persist in individuals during transition from a milk-based to a more diversified diet. Here, we investigated adaptation of Bifidobacterium longum to the changing nutritional environment. Genomic characterization of 75 strains isolated from nine either exclusively breast- or formula-fed (pre-weaning) infants in their first 18 months revealed subspecies- and strain-specific intra-individual genomic diversity with respect to carbohydrate metabolism, which corresponded to different dietary stages. Complementary phenotypic studies indicated strain-specific differences in utilization of human milk oligosaccharides and plant carbohydrates, whereas proteomic profiling identified gene clusters involved in metabolism of selected carbohydrates. Our results indicate a strong link between infant diet and B. longum diversity and provide additional insights into possible competitive advantage mechanisms of this Bifidobacterium species and its persistence in a single host.