The apical sodium-dependent bile acid transporter (ASBT) is the primary transporter for the uptake of bile acids in the small intestine. It is localized on the apical membrane of the ileal enterocytes and is known for its high capacity and affinity for taurocholic acid in vitro. However, less is known about its activity towards taurocholic acid and other bile acids in vivo due to the lack of a suitable model. The aim of this study was to validate precision-cut intestinal slices (PCIS) of rat and human intestine as ex vivo model to study the activity of ASBT-mediated transport, and subsequently to study regional and interspecies differences in ASBT function. PCIS maintain the natural cell polarization and communication, expression of transporters and metabolizing enzymes. Therefore this model is expected to be more relevant to the in vivo situation than in vitro cell culture models. PCIS of human and rat ileum were prepared and incubated with 0.04–2.00 mM taurocholic acid, deoxycholic acid and cholic acid, respectively at 4 and 37 °C. In this study, the respective contribution of active uptake appeared to be higher for taurocholic acid whereas the passive diffusion was higher for deoxycholic acid and cholic acid. Furthermore, the rank order of calculated (apparent) Km and Vmax for these bile acids is in line with literature reports. The active uptake of taurocholic acid in rat PCIS could be inhibited partly by simvastatin and fluvastatin and was fully inhibited by the specific inhibitor GSK2299027B, supporting the involvement of rat ASBT as uptake transporter. In both species, the ASBT-mediated active uptake of bile acids was observed only in the ileum whereas only passive diffusion was observed in the jejunum and colon. In addition, ASBT activity was higher in the human ileum compared to the rat ileum. In the future rat and human PCIS can be used to study the uptake of new ASBT substrates and as a screening method for potential ASBT inhibitors.