Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes.