A toxicogenomics approach was used to qualitatively and quantitatively compare the gene expression changes in human and rat primary hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes from five individual rats and five individual humans were exposed for 24 h to 11 concentrations of TCDD ranging from 0.00001 to 100nM and a vehicle control. Gene expression changes were analyzed using whole-genome microarrays containing 13,002 orthologs. Significant changes in expression of individual orthologs at any concentration (fold change [FC] ± 1.5 and false discovery rate < 0.05) were higher in the rat (1547) compared with human hepatocytes (475). Only 158 differentially expressed orthologs were common between rats and humans. Enrichment analysis was performed on the differentially expressed orthologs in each species with 49 and 34 enriched human and rat pathways, respectively. Only 12 enriched pathways were shared between the two species. The results demonstrate significant cross-species differences in expression at both the gene and pathway level. Benchmark dose analysis of gene expression changes showed an average 18-fold cross-species difference in potency among differentially expressed orthologs with the rat more sensitive than the human. Similar cross-species differences in potency were observed for signaling pathways. Using the maximum FC in gene expression as a measure of efficacy, the human hepatocytes showed on average a 20% lower efficacy among the individual orthologs showing differential expression. The results provide evidence for divergent cross-species gene expression changes in response to TCDD and are consistent with epidemiological and clinical evidence showing humans to be less sensitive to TCDD-induced hepatotoxicity.