1. During the treatment of neonatal apnea, formula-fed infants, compared to breastfed infants, show nearly three-fold increase in clearance of caffeine, a substrate of cytochrome P450 1A (CYP1A) and in part CYP3A4. However, human milk is known to contain higher concentrations of environmental pollutants than infant formula, which are potent CYP1A inducers. To gain insight into the mechanism underlying this apparent contradiction, we characterized CYP1A and CYP3A4 induction by human milk and cow milk-based infant formula. 2. The mRNA and protein expression of CYP1A1/1A2 were significantly induced by cow milk-based formula, but not by human milk, in HepG2 cells. 3. Luciferase reporter assay demonstrated that cow milk-based formula but not human milk activated aryl hydrocarbon receptor (AhR) significantly. The cotreatment of 3,4-dimethoxyflavone, an AhR antagonist, abolished the formula-induced CYP1A expression. In addition, AhR activation by dibenzo[a,h]anthracene, a potent AhR agonist, was significantly suppressed by infant formula and even more by human milk. 4. In contrast, CYP3A4 mRNA expression was only mildly induced by formula and human milk. Consistently, neither formula nor human milk substantially activated pregnane X receptor (PXR). 5. Effects of whey and soy protein-based formulas on the AhR–CYP1A and the PXR–CYP3A4 pathways were similar to those of cow milk-based formula. 6. In conclusion, infant formula, but not human milk, enhances in vitro CYP1A expression via an AhR-mediated pathway, providing a potential mechanistic basis for the increased caffeine elimination in formula-fed infants.