The aim of this study was to investigate the prebiotic activities of dextran (LM742) produced by Leuconostoc mesenteroides SPCL742 in the aspect of the human gut microbial ecosystem focusing on microbiome and metabolome changes in in vitro colonic fermentation. LM742 dextran had a medium-chain structure with the molecular weight of 1394.87 kDa (DP = 7759.22) and α-1,6 and α-1,3 linkages with a 26.11 : 1 ratio. The LM742 dextran was resistent to digestive enzymes in the human gastrointestinal conditions. The individual cultivation of 30 intestinal bacteria with LM742 dextran showed the growth of Bacteroides spp., whereas in vitro human fecal fermentation with LM742 exhibited the symbiotic growth of Bacteroides spp. and beneficial bacteria such as Bifidobacterium spp. Further co-cultivation of Bacteroides xylanisolvens and several probiotics indicated that B. xylanisolvens provides a cross-feeding of dextran to probiotics. In fecal fermentation, LM742 dextran resulted in increased concentrations of short-chain fatty acids, valerate and pantothenate, but it rarely affected the conversion of betaine to trimethylamine. Lastly, LM742 dextran inhibited the adhesion of pathogenic E. coli to human epithelial cells. Taken together, these results demonstrate the prebiotic potential of LM742 dextran as a health-beneficial polysaccharide in the human intestine.