Human milk contains prebiotic oligosaccharides which stimulate the growth of intestinal bifidobacteria and lactobacilli. It is unclear whether the prebiotic capacity of human milk contributes to the larger bile salt pool size and the more efficient fat absorption in infants fed human milk compared to formula. We determined the effect of prebiotic oligosaccharides on bile salt metabolism in rats. Rats were fed a control diet, or an isocaloric diet containing a mixture of galactooligosaccharides (GOS), long chain fructooligosaccharides (lcFOS) and acidified oligosaccharides (AOS) for three weeks. We determined synthesis rate, pool size, and fractional turnover rate (FTR) of the primary bile salt cholate using stable isotope dilution methodology. We quantified bile flow and biliary bile salt secretion rates through bile canulation. Prebiotic intervention resulted in significant changes in fecal and colonic flora: the proportion of Lactobacilli increased with 344% (p<0.01) in colon content and with 139% (p<0.01) in feces compared to the control group. The number of Bifidobacteria also increased with 366% (p<0.01) in colon content and with 282% in feces after the prebiotic treatment. Furthermore, pH in both colon and feces decreased significantly with respectively 1.0 and 0.5 pH point. However, despite this alteration of intestinal bacterial flora, no significant effect on relevant parameters of bile salt metabolism and cholate kinetics was found. The present data in rats do not support the hypothesis that prebiotics naturally present in human milk contribute to a larger bile salt pool size or altered bile salt pool kinetics.