Background: With the rise of antibiotic resistance, new alternatives are being sought to effectively modulate the characteristics of gut microbiota to obtain pathogen resistance without the use of antibiotics. In the past, an oligosaccharide derivative of carrots, galursan HF 7K (GHF7K), has been used clinically in Austria and recently in the fowl-industry to promote health. This study examined the potential role of GHF7K as a prebiotic to alter the gut microbiota in mice. Methods: Mice were fed either a control diet (CT) or a diet containing 2% GHF7K in the water and chow for 2 weeks, and weight, food and water consumption, gut microbiota and ion composition of the intestinal fluid were examined. Results: Dietary supplement of GHF7K did not alter mouse weight or daily food consumption. Additionally, no changes were observed in the total number of luminal or mucosa-associated bacteria populations in GHF7K-fed mice. GHF7K supplementation significantly altered the composition of luminal, and to a less extent, mucosa-associated bacterial populations at the level of the phyla, with region-specific differences. Similar to antibiotic use, Proteobacteria number was increased in the ileum and colon of GHF7K-fed mice, with no changes in the number of beneficial Lactobacillus and Bifidobacterium genera of phylum Firmicutes. Corresponding with the altered gut microbiota, changes in the ion composition of the intestinal fluid were observed. An increased Cl- concentration was observed in the duodenum and jejunum, while the Na+ concentration was increased in the cecum of GHF7K-fed mice. Decreases were observed in the K+ concentration in the cecum and distal colon. Conclusions: Dietary supplement of GHF7K is capable of altering the gut microbiota, which correlates to changes in the intestinal environment. These data suggest that GHF7K dietary supplement can purposefully be used to alter the gut microbiota, and thus could potentially represent an alternative approach to prophylactic antibiotic use.