To obtain new lactose-derived prebiotic, epilactose and its derivative epilactitol were synthesised with high purity (>98%), and their structural characteristics were investigated. To determine the prebiotic potential of epilactose and epilactitol, these two compounds were fermented in vitro with human faecal microbiota. Consequently, epilactose addition increased the relative abundance of Bifidobacterium, Megamonas, Blautia, and Phascolarctobacterium, whereas epilactitol addition enhanced the relative abundance of Anaerostipes, Megamonas, Blautia and Phascolarctobacterium after in vitro fermentation for 24 h. The two compounds increased the production of total amount of short-chain fatty acids (SCFAs); in particular, epilactose addition increased acetate and n-butyrate production (P < 0.05), and epilactitol addition increased propionate and n-butyrate production (P < 0.05) after in vitro fermentation for 24 h. In conclusion, epilactose and epilactitol can be utilised by human faecal microbiota, modify microbial community, increase SCFA production, they may serve as novel prebiotics to bring health benefits for humans.(c) 2023 Elsevier Ltd. All rights reserved.