Adenosine triphosphate (ATP)-binding cassette, sub-family B, member 4 (ABCB4), also called multidrug resistance 3 (MDR3), is a member of the ATP-binding cassette transporter superfamily, which is localized at the canalicular membrane of hepatocytes, and mediates the translocation of phosphatidylcholine into bile. Phosphatidylcholine secretion is crucial to ensure solubilization of cholesterol into mixed micelles and to prevent bile acid toxicity towards hepatobiliary epithelia. Genetic defects of ABCB4 may cause progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare autosomic recessive disease occurring early in childhood that may be lethal in the absence of liver transplantation, and other cholestatic or cholelithiasic diseases in heterozygous adults. Development of therapies for these conditions requires understanding of the biology of this transporter and how gene variations may cause disease. This review focuses on our current knowledge on the regulation of ABCB4 expression, trafficking and function, and presents recent advances in fundamental research with promising therapeutic perspectives.