Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug–drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50= 0.3 µM) compared to rifampicin (IC50= 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin’s concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00204-023-03531-2.