Deficiency of ABCB4 is associated with several forms of cholestasis in humans. Abcb4^-/- mice also develop cholestasis, but it remains uncertain what role other canalicular transporters play in the development of this disease. We examined the expression of these transporters in Abcb4^-/- mice, compared to their wild-type littermate controls at ages of 10 days, 3, 6 and 12 weeks. Elevated plasma bile acid levels were already detected at 10 days and at all ages thereafter in Abcb4^-/- mice. The expression of Bsep, Mrp2, Atp8b1, Abcg5 and Abcg8 liver proteins did not change at 10 days, but Bsep, Mrp2, and Atp8b1 were reduced, while Abcg5 and Abcg8 expression were increased in Abcb4^-/- mice at all later ages. Lower bile acid concentrations were also detected in the bile of 6 weeks Abcb4^-/- mice. Immunofluorescence labeling revealed distorted canalicular architecture in the liver tissue by 12 weeks in Abcb4^-/- mice. While Bsep and Mrp2 remained associated with the apical membrane, Atp8b1 was now localized in discrete punctuate structures adjacent to the canalicular membrane in these mice. Expression of Bsep mRNA was increased in the livers of 10 day old Abcb4^-/- mice, whereas Ostα was decreased. By 12 weeks, Bsep, Mrp2, and Abcg5 mRNA were all increased, while Ostα and Ntcp were reduced. These findings indicate that canalicular transporters that determine the formation of bile are altered early in the development of cholestasis in Abcb4^-/- mice and may contribute to the pathogenesis of cholestasis in this disorder.