Estradiol 17b-D-glucuronide (E 2 17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E 2 17G-induced cholestasis. E 2 17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E 2 17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E 2 17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Go ¨6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E 2 17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E 2 17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [ 3 H]taurocholate and glutathione until the end of the perfusion period. Unlike Go ¨6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. Conclusion: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E 2 17G through sustained internalization of canalicular transporters endocytosed via cPKC. (HEPATOLOGY 2010;52:1465-1476) B ile formation is a highly regulated process. It depends on the coordinated action of a number of transporters in the sinusoidal and canalicular domains of hepatocytes. The transfer of solutes across the canalicular membrane is the rate-limiting step in bile formation; therefore, functional alterations in canalicular transporters are more likely to impair bile flow generation. Bile salt export pump (Bsep; adenosine triphosphate-binding cassette b11) and multidrug resistance-associated protein 2 (Mrp2;