Existing prebiotics, such as fructo-oligosaccharides (FOSs), can be modified to enhance their functionality or introduce additional functionalities. This study aimed to investigate the fermentation characteristics and prebiotic potential of enzymatically synthesized butyryl-FOSs. The esters were successfully synthesized through the reaction of butyric acid and FOSs using both chemical and enzymatic methods, denoted as A-FOSs and B-FOSs, respectively, for comparative analysis. The esterification degree of each component in A-FOSs was significantly higher than that of B-FOSs. Subsequently, the obtained esters were characterized for their fermentation properties, degradation mode and potential prebiotic effects using an in vitro simulated colonic fermentation model. Enzymes of human gut microbiota were found to preferentially cleave the glycosidic bond to the unit without butyryl group and release the sugars for utilization. A significant increase in butyric acid levels was observed during fermentation after the supplementation of B-FOSs. The 16S rRNA gene sequencing, absolute quantification of microbiota, and selected probiotic strains culture showed that B-FOSs supplementation promoted the growth of beneficial bacteria while reducing harmful ones. These results suggest that B-FOSs hold promise as novel prebiotics, possessing dual functions of modulating gut microbiota and delivering butyric acid to the colon in a targeted manner, ultimately contributing to improved gut health.