Cytochrome P450 3A4 (CYP3A4) is a member of the CYP family and is an important enzyme in drug metabolism. A compound that inhibits CYP3A4 activity could also affect the pharmacokinetics of other substrates, resulting in drug–drug interactions (DDIs) that could cause side effects. Pharmacokinetic data from drug-development studies in rats often determine the dosage used in human clinical trials. It is therefore useful to understand differences in metabolism in different species at an early stage in drug development. Human and rat CYP3A enzymes show different inhibition profiles with different drugs, although the mechanisms involved are not yet clear. Here we built three-dimensional quantitative structure–activity relationship (3D-QSAR) models using structure-based comparative molecular field analysis (CoMFA), to predict the direct inhibitory activity of ligands for human CYP3A4 and rat CYP3A1, based on computer-ligand docking. The alignment of the ligand docking poses suggested that key amino acid–ligand interactions (e.g., Thr309 in CYP3A4 and Pro310 in CYP3A1) characterized the different potencies with which the ligands inhibited CYP3A4 and CYP3A1. The 3D-QSAR models for human and rat CYP3A family inhibitors predicted the potency of inhibitors and could be useful for assessing DDIs at an early stage in drug discovery.