A recent study revealed that the accumulation of gut microbiota-produced acetate (GMPA) led to insulin over-secretion and obesity symptom. To further develop this scientific point, the effect of resistant starch (RS) or exogenous acetate carried by RS (RSA) in the gut on metabolic syndrome is investigated using diet-induced obese rats. Methods and results: The metabonomics analysis shows that the gut of rats in the RSA group generate more butyrate in both serum and feces rather than acetate compared to the rats in RS group, indicating the conversion among metabolites, in particular from acetate to butyrate via gut microbiota. Consistently, the gut microbiome uses acetate as a substrate to produce butyrate, such as Coprococcus, Faecalibacterium, Roseburia, and Eubacterium and was highly promoted in RSA group, which further supports the metabolic conversion. This is the first report to reveal the accumulation of gut microbiota-produced butyrate (GMPB) but not GMPA significantly enriched AMPK signaling pathway with reduced expression of lipogenesis-associated genes for suppressing sphingosines and ceramides biosynthesis to trigger insulin sensitivity. Conclusion: Gut microbiome profile and lipogenesis pathway are regulated by GMPB, which substantially influences energy harvesting in the gut from patterns opposed to GMPA.