Background and purpose Dietary fiber has been used to prevent obesity by regulating the intestinal flora and promoting the production of short-chain fatty acids (SCFAs). However, it is insufficient to conclude the decisive role of microbiota and SCFAs by adding oligosaccharides to foods without caloric balance. Experimental approach The effects of oligosaccharides and their regulated microflora on the development of obesity in mice were studied by designing a high-fat diet with equal calories but different contents of oligosaccharides. Simulation of SCFAs produced by intestinal flora to explore the regulatory mechanisms of bacterial flora in hormone and lipid synthesis. Key results Isocaloric diets demonstrated that appropriate rather than excess oligosaccharides prevent obesity by regulating appetite. Such an appetite was inhibited by oligosaccharides but promoted by SCFAs. Furthermore, promoted appetite was tightly related to decreased insulin and increased acyl-CoA binding protein, which was correlated with SCFA-induced fat degradation. Interestingly, drinking butyrate alleviated obesity even with higher calorie intake. Molecular docking demonstrated that conversion of butyrate to butyryl-CoA converted from butyrate, as a structural analog of acetyl-CoA, inhibits the activity of acetyl-CoA carboxylase. Conclusion and implications Together, these findings demonstrate the limitation of fermentable fiber and provide possible obesity therapeutic targets that inhibit bacterial fermentation or increase the ratio of butyrate/acetate.