Key points The short‐chain fatty acids (SCFAs) are bacterial metabolites produced during the colonic fermentation of undigested carbohydrates, such as dietary fibre and prebiotics, and can mediate the interaction between the diet, the microbiota and the host. We quantified the fraction of colonic administered SCFAs that could be recovered in the systemic circulation, the fraction that was excreted via the breath and urine, and the fraction that was used as a precursor for glucose, cholesterol and fatty acids. This information is essential for understanding the molecular mechanisms by which SCFAs beneficially affect physiological functions such as glucose and lipid metabolism and immune function. Abstract The short‐chain fatty acids (SCFAs), acetate, propionate and butyrate, are bacterial metabolites that mediate the interaction between the diet, the microbiota and the host. In the present study, the systemic availability of SCFAs and their incorporation into biologically relevant molecules was quantified. Known amounts of 13C‐labelled acetate, propionate and butyrate were introduced in the colon of 12 healthy subjects using colon delivery capsules and plasma levels of 13C‐SCFAs 13C‐glucose, 13C‐cholesterol and 13C‐fatty acids were measured. The butyrate‐producing capacity of the intestinal microbiota was also quantified. Systemic availability of colonic‐administered acetate, propionate and butyrate was 36%, 9% and 2%, respectively. Conversion of acetate into butyrate (24%) was the most prevalent interconversion by the colonic microbiota and was not related to the butyrate‐producing capacity in the faecal samples. Less than 1% of administered acetate was incorporated into cholesterol and <15% in fatty acids. On average, 6% of colonic propionate was incorporated into glucose. The SCFAs were mainly excreted via the lungs after oxidation to 13CO2, whereas less than 0.05% of the SCFAs were excreted into urine. These results will allow future evaluation and quantification of SCFA production from 13C‐labelled fibres in the human colon by measurement of 13C‐labelled SCFA concentrations in blood.