There is increasing interest in identifying novel dietary nondigestible carbohydrates capable of modulating the composition and/or metabolic activities of the gut microbiota. This work assessed the differential modulatory influence of novel galacto-oligosaccharides derived from lactulose (GOS-Lu) in comparison with commercial galacto-oligosaccharides derived from lactose (GOS-La) in gut microbiota of growing rats (5 weeks old). Rats were fed either a control diet or diets containing 1% (w/w) of GOS-Lu or GOS-La, and cecal and colonic contents were collected after 14 days of treatment. Compared to controls, GOS-Lu had significantly more bifidobacteria within the large intestine, showing a significant and selective increase of Bifidobacterium animalis in the cecum and colon; however, no significant differences in the number of bifidobacteria among GOS-Lu and GOS-La groups were observed. Both types of GOS significantly increased the number of the Eubacterium rectale/Clostridium coccoides group. These findings support a prebiotic role of galactosyl-fructoses in functional foods.