Transient latent cholestasis in young rats born from mothers with obstructive cholestasis during pregnancy (OCP) has been reported. The cause of this congenital impairment and the long-term effect on the pups of treating their mothers with ursodeoxycholic acid (UDCA) during pregnancy were investigated. Complete biliary obstruction was imposed on day 14 of pregnancy and UDCA treatment was begun on day 15. Serum bile acids (BAs) concentrations were elevated in 4-week-old pups born from OCP, but not OCP + UDCA, mothers. However, gas chromatographic/mass spectrometric analysis of BA species in basal bile indicated the presence of significant differences among all experimental groups (control, OCP, and OCP + UDCA). Canalicular plasma membrane fluidity was reduced in OCP, but not in OCP + UDCA, pups. Screening by reverse transcription followed by real-time quantitative polymerase chain reaction of the steady-state levels of mRNA of genes related to hepatobiliary function revealed changes (upregulation of Cyp7a1 and Mrp1 and down-regulation of Abcg5 and Abcg8) in OCP group, which were prevented by UDCA treatment. Electron microscopy examination showed multilamellar bodies occupying part of the canalicular lumen in OCP pups. Their number and size were reduced in animals born from OCP + UDCA mothers. In OCP, but not OCP + UDCA, the stimulation of bile flow and BA output induced by taurocholate administration were reduced and cholesterol/BA output ratio was increased, whereas phospholipid/BA output ratio was enhanced in both groups (OCP > OCP + UDCA). In conclusion, UDCA treatment of rats with cholestasis during pregnancy has long-term beneficial effects on their offspring by preventing in part the congenital impairment in hepatobiliary function of the pups that affects their biliary lipid secretion.