Dietary glycerol supplementation in aquaculture feed is seen as an alternative and inexpensive way to fuel fish metabolism, attenuate metabolic utilization of dietary proteins and, subsequently, reduce nitrogen excretion. In this study, we evaluated the impact of dietary glycerol supplementation on nitrogen excretion of European seabass (Dicentrarchus labrax) and its effects on metabolite profile and bacterial community composition of gut digesta. These effects were evaluated in a 60-day trial with fish fed diets supplemented with 2.5% or 5% (w/w) refined glycerol and without glycerol supplementation. Nuclear magnetic resonance spectroscopy and high-throughput 16S rRNA gene sequencing were used to characterize the effects of glycerol supplementation on digesta metabolite and bacterial community composition of 6-h postprandial fish. Our results showed that ammonia excretion was not altered by dietary glycerol supplementation, and the highest glycerol dosage was associated with significant increases in amino acids and a decrease of ergogenic creatine in digesta metabolome. Concomitantly, significant decreases in putative amino acid degradation pathways were detected in the predicted metagenome analysis, suggesting a metabolic shift. Taxon-specific analysis revealed significant increases in abundance of some specific genera (e.g.,BurkholderiaandVibrio) and bacterial diversity. Overall, our results indicate glycerol supplementation may decrease amino acid catabolism without adversely affecting fish gut bacterial communities. Key points center dot Glycerol can be an inexpensive and energetic alternative in fish feed formulations. center dot Glycerol did not affect nitrogen excretion and gut bacteriome composition. center dot Glycerol reduced uptake of amino acids and increased uptake of ergogenic creatine. center dot Glycerol reduced putative amino acid degradation pathways in predicted metagenome.