Estrogen-induced intrahepatic cholestasis (EIC) has increased incidence during pregnancy, and within women taking oral contraception and postmenopausal hormone replacement therapy. However, the pathology underlying EIC is not well understood. The aim of the present study was to identify key pathways and candidate genes in estrogen-induced intrahepatic cholestasis (EIC) that may be potential targets for diagnosis and treatment. A whole-genome microarray (4x44K) analysis of a 17 alpha-ethinylestradiol (EE)-induced EIC rat liver model was performed. Bioinformatics-based methods were used to identify key pathways and candidate genes associated with EIC. The candidate genes were validated using a reverse transcription quantitative polymerase chain reaction assay. A total of 455 genes were differentially expressed (P<0.05 and fold change >2.0) following EE treatment, including 225 downregulated genes and 230 upregulated genes. Sulfotransferase family 1E member 1, cytochrome P450 family 3 subfamily A member 2, carbonic anhydrase 3, leukotriene C4 synthase and ADAM metallopeptidase domain 8 were the 5 candidate genes identified to be differentially expressed and involved in the metabolism of estrogens and bile acids and the regulation of inflammation and oxidative stress. The Analyses of Gene Ontology enrichment, Kyoto Encyclopedia of Genes and Genomes pathways and protein-protein interaction network associated-modules identified several key pathways involved in the homeostasis of lipids and bile acids and in AMPK, p53 and Wnt signaling. These key pathways and candidate genes may have critical roles in the pathogenesis of EIC. In addition, reversing the abnormal expression of candidate genes or restoring the dysfunction of key pathways may provide therapeutic opportunities for patients with EIC.