During pregnancy, proper hepatobiliary transport and bile acid synthesis protect the liver from cholestatic injury and regulate the maternal and fetal exposure to bile acids, drugs, and environmental chemicals. The objective of this study was to determine the temporal messenger RNA (mRNA) and protein profiles of uptake and efflux transporters as well as bile acid synthetic and conjugating enzymes in livers from virgin and pregnant mice on gestational days (GD) 7, 11, 14, and 17 and postnatal days (PND) 1, 15, and 30. Compared with virgins, the mRNAs of most transporters were reduced approximately 50% in pregnant dams between GD11 and 17. Western blot and immunofluorescence staining confirmed the downregulation of Mrp3, 6, Bsep, and Ntcp proteins. One day after parturition, the mRNAs of many uptake and efflux hepatobiliary transporters remained low in pregnant mice. By PND30, the mRNAs of all transporters returned to virgin levels. mRNAs of the bile acid synthetic enzymes in the classic pathway, Cyp7a1 and 8b1, increased in pregnant mice, whereas mRNA and protein expression of enzymes in the alternative pathway of bile acid synthesis (Cyp27a1 and 39a1) and conjugating enzymes (Bal and Baat) decreased. Profiles of transporter and bile acid metabolism genes likely result from coordinated downregulation of transcription factor mRNA (CAR, LXR, PXR, PPARα, FXR) in pregnant mice on GD14 and 17. In conclusion, pregnancy caused a global downregulation of most hepatic transporters, which began as early as GD7 for some genes and was maximal by GD14 and 17, and was inversely related to increasing concentrations of circulating 17β-estradiol and progesterone as pregnancy progressed.