Fructo-oligosaccharides (FOS) have well-known bifidogenic effects as probiotics. In this study, esterification was adopted for FOS modification to produce better prebiotic properties. We synthesized and characterized acetylated fructo-oligosaccharides (Ac-FOS) and butyrylated fructo-oligosaccharides (Bu-FOS) as candidate prebiotics. Antioxidant activity and prebiotic esactiviti were evaluated as important indicators. We found, surprisingly, that butyrylation was an effective method in significantly improving the antioxidant activity of FOS. The fermentation products of feces from mice added to Ac-FOS and Bu-FOS, were investigated in vitro, including changes of pH values, short-chain fatty acids (SCFAs) production, and microbiota composition. Supplementation of Ac-FOS or Bu-FOS increased pH values and promoted the growth and activity of beneficial intestinal bacteria, such as Bifidobacteria and Lactobacillus. More importantly, the levels of prebiotic SCFAs were obviously elevated as detected by Gas Chromatography–Mass Spectrometry (GC-MS). Results suggest that Ac-FOS and Bu-FOS have great potential applications in SCFA delivery systems and gut microbiota regulation.