Rat hepatocytes express aquaporin-9 (AQP9), a basolateral channel permeable to water, glycerol and other small neutral solutes. While liver AQP9 is known for mediating the uptake of sinusoidal blood glycerol its relevance in bile secretion physiology and pathophysiology remains elusive. Here, we evaluated whether defective expression of AQP9 is associated to secretory dysfunction of rat hepatocytes following bile duct ligation (BDL). By immunoblotting, one-day BDL resulted in a slight decrease of AQP9 protein in basolateral membranes and a simultaneous increase of AQP9 in intracellular membranes. This pattern was steadily accentuated in the subsequent days of BDL as at 7-days BDL basolateral membrane AQP9 decreased by 85% whereas intracellular AQP9 increased by 115%. However, the AQP9 immunoreactivity of the total liver membranes from day 7 of BDL rats was reduced by 49% compared to the sham counterpart. Results were confirmed by immunofluorescence and immunogold electron microscopy and consistent with biophysical studies showing considerable decrease of the basolateral membrane osmotic water permeability of cholestatic hepatocytes. The AQP9 mRNA was slightly reduced only at day 7 of BDL indicating that the AQP9 dysregulation was mainly occurring at a posttranslational level. The altered expression of liver AQP9 during BDL was not dependent on insulin, a hormone known to regulate negatively AQP9 at a transcriptional level, since insulinemia was unchanged in 7-days BDL rats. Overall, these results suggest that extrahepatic cholestasis leads to downregulation of AQP9 in the hepatocyte basolateral plasma membrane and dysregulated aquaporin channels contribute to bile flow dysfunction of cholestatic hepatocyte.