The aim of this research was to investigate the impact of a diet containing galactooligosaccharide (GOS)-fish peptide (FP) conjugates prepared via Maillard reaction on the colonic fermentation properties and the composition of gut microbiota in Sprague-Dawley rats. The rats were fed the GOS diet, FP diet, GOS and FP mixture (GOS/FP) diet, GOS glycated with FP (80 °C for 120 min, G-GOS/FP) diet, or control (CK) diet for three weeks. Compared to the GOS/FP diet, the G-GOS/FP diet greatly changed the pattern of SCFA production in the hindgut of rats, by increasing the total SCFA (44%), butyrate (55%) and propionate (1.23-fold) levels in the proximal colon, and the butyrate levels (74%) in the distal colon, and decreased the production of ammonia in feces (P < 0.05). The G-GOS/FP altered the colonic microbiota by increasing (P < 0.05) the relative abundance of Anaerovibrio (7.43-fold) and Prevotella-9 (2.47-fold), and by decreasing (P < 0.05) the relative abundance of Alloprevotella (0.57-fold) and Holdemanella (0.64-fold), and showed a similar relative abundance of Bifidobacterium, when compared with GOS/FP. The GOS/FP diet increased the number of Lactobacillus and the intensity of fermentation in the cecum, but the G-GOS/FP diet and GOS diet did not have these effects, showing that the glycation clearly altered the fermentability of the fish peptide. It is concluded that the glycation-induced modification of GOS by mild thermal treatments showed its fermentation persistence in the colon of the host, and improved some prebiotic activities of GOS. These results may provide new strategies for oligosaccharides in combination with peptides to modulate the intestinal environment to promote human health.