Acetate, propionate and butyrate are the main short-chain fatty acids (SCFAs) that arise from the fermentation of fibers by the colonic microbiota. While many studies focus on the regulatory role of SCFAs, their quantitative role as a catabolic or anabolic substrate for the host has received relatively little attention. To investigate this aspect, we infused conscious mice with physiological quantities of stable isotopes [1-13C]acetate, [2-13C]propionate or [2,4-13C2]butyrate directly into the cecum, which is the natural production site in mice, and analyzed their interconversion by the microbiota as well as their metabolism by the host. Cecal interconversion - pointing to microbial cross-feeding - was high between acetate and butyrate, low between butyrate and propionate and almost absent between acetate and propionate. As much as 62% of infused propionate was used in whole-body glucose production, in line with its role as gluconeogenic substrate. Conversely, glucose synthesis from propionate accounted for 69% of total glucose production. The synthesis of palmitate and cholesterol in the liver was high from cecal acetate (2.8% and 0.7%, respectively) and butyrate (2.7% and 0.9%, respectively) as substrates, but low or absent from propionate (0.6% and 0.0%, respectively). Label incorporation due to chain elongation of stearate was approximately 8-fold higher than de novo synthesis of stearate. Microarray data suggested that SCFAs exert only a mild regulatory effect on the expression of genes involved in hepatic metabolic pathways during the 6h infusion period. Altogether, gut-derived acetate, propionate and butyrate play important roles as substrates for glucose, cholesterol and lipid metabolism.