P-glycoprotein (P-gp) the multidrug transporter is a well-characterised member of the super-family of ATP-binding cassette (ABC) transporters, and mediates the clearance of xenotoxins against steep concentration gradients at the expense of ATP hydrolysis. The primary function of this protein is to prevent the uptake of toxic compounds from the gut into the body, and to protect vital structures such as the brain, cerebrospinal fluid, testis, foetus and bone marrow against toxins. Although P-gp transports a wide range of compounds, which is advantageous, it can also be a disadvantage and may interfere with the delivery of drugs to target tissues resulting in multidrug resistance. In the present review: (i) we consider our current understanding of the structure of P-glycoprotein, (ii) discuss substrate binding and its coupling to ATPase activity, (iii) provide insight into key features which define P-glycoprotein substrates/inhibitors and the ability to predict potential substrates in silico, (iv) provide an overview of existing models of pump function and (v) present emerging concepts into the regulation of P-glycoprotein expression, with particular reference to multidrug resistance.