In order to elaborate on the impact of amoxicillin treatment on the in vitro fermentation of specific structures of galacto-oligosaccharides (GOS), GOS was fractionated based on its degree of polymerization (DP) and the fermentation of individual DPs was studied. Different DP fractions of GOS and different isomeric structure within a DP fraction were preferentially degraded depending on the treatment applied to the microbiota. For the non-treated microbiota, the small DP fractions (dimers and trimers) were preferentially degraded as compared to the large DP fractions (tetramers till hexamers). ß-d-Gal-(1¿4/6)-d-Glc and ß-d-Gal-(1¿4)-ß-d-Gal-(1¿4)-d-Glc were the isomeric structures preferentially degraded within the DP2 and DP3 fraction, respectively. The fermentation of each size-fraction induced the production of various short chain fatty acids and the growth of several species of bifidobacteria. For amoxicillin-treated microbiota, the large size-fractions of GOS were preferentially degraded as compared to the small fractions. ß-d-Gal-(1¿4)-d-Gal and ß-d-Gal-(1¿4)-ß-d-Gal-(1¿3)-d-Glc were the isomeric structures preferentially degraded within the DP2 and DP3 fraction, respectively. Butyrate was only produced upon the fermentation of the large size-fractions. The differences in metabolic pattern of GOS depending on the treatment applied correlated with the changes in the microbiota composition, especially the growth of bifidobacteria. These results suggest that GOS, especially its large size-fractions, support the recovery of bifidobacteria and butyrate-producing bacteria after a treatment of the microbiota with amoxicillin.