Sandwich-cultured primary rat hepatocytes are often used as an in vitro model in toxicology and pharmacology. Loss of liver specific functions, in particular the decline of cytochrome P450 (CYP450) enzyme activity, however, limits the value of this model for prediction of in vivo toxicity. In this study, we investigated whether a hepatic in vitro system with improved metabolic competence enhances the predictability for coumarin-induced in vivo toxicity by using a toxicogenomics approach. Therefore, primary rat hepatocytes were cultured in sandwich configuration in medium containing a mixture of low concentrations of CYP450 inducers, phenobarbital, dexamethasone, and beta-naphthoflavone. A toxicogenomics approach was employed enabling comparison of similar mechanistic endpoints at the molecular level between in vitro and in vivo, namely compound-induced changes in multiple genes and signaling pathways. Toxicant-induced cytotoxic effects and gene expression profiles observed in hepatocytes cultured in modified medium and hepatocytes cultured in standard medium (without inducers) were compared to results from a rat in vivo study. Coumarin was used as a model compound because its toxicity depends on bioactivation by CYP450 enzymes. Metabolism of coumarin towards active metabolites, coumarin-induced cytotoxicity, and gene expression modulation were more pronounced in hepatocytes cultured in modified medium compared to hepatocytes cultured in standard medium. Additionally, more genes and biological pathways were similarly affected by coumarin in hepatocytes cultured in modified medium and in vivo. In conclusion, these experiments showed that for coumarin-induced toxicity sandwich-cultured hepatocytes maintained in modified medium better represent the situation in vivo compared to hepatocytes cultured in standard medium.