The hepatocarcinoma (HepG2) and colorectal adenocarcinoma (Caco-2) cell lines are widely used for pharmacokinetic and pharmacodynamic modeling of xenobiotic metabolism in human liver and intestine. Phenol red is commonly contained in cell culture medium as a pH indicator, however, the impact of phenol red on the expression of metabolic and transporter associated genes in cell models is limited. This study examined the effect of phenol red on the rifampicin-induced expression of four cytochrome P450 (CYP) enzymes (CYP1A2, CYP2E1, CYP3A4, and CYP3A5), the solute carrier organic anion transporter 1B1 (SLCO1B1), and three nuclear receptors related to the regulation of metabolism (pregnane X receptor PXR, aryl hydrocarbon receptor AhR, and constitutive androstane receptor CAR) in HepG2 and Caco-2 cells. HepG2 (1.25×105 cells/well) and Caco-2 (5×104 cells/well) were cultured in phenol-red containing medium in 24-well plates for 72 hours before induction with rifampicin (5 µM) in phenol red-containing or phenol red-free media for 72 hours. Total RNA was reverse transcribed and expression of the target genes was determined by RT/qPCR. Phenol red suppressed induction of CYP3A4, CYP3A5, and AhR mRNA by rifampicin in both HepG2 and Caco-2 cells, and suppressed rifampicin-induced CYP1A2 expression in HepG2 cells and rifampicin-induced expression of CYP2E1 and SLCO1B1 in Caco-2 cells. Rifampicin did not induce expression of the PXR and CAR nuclear receptors in either medium. The different effects of phenol red on the regulation of CYP1A2, CYP2E1, and SLCO1B1 in HepG2 and Caco-2 cell lines is therefore an important factor to consider when using these cell lines for pharmacokinetic and pharmacodynamic modeling of xenobiotics.