The HepaRG cell line is a well-differentiated human hepatoma cell line proposed as a surrogate for human hepatocytes, especially for hepatic detoxification studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, which represents a key hallmark of hepato-biliary drug transport, remains however incompletely documented in HepaRG cells. The present study was therefore designed to analyze transporter location in HepaRG cells, which exhibit mRNA expressions of most of hepatic transporters. HepaRG cells were demonstrated, through immunofluorescence staining, to express several drug transporters at their sinusoidal pole, especially the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of HepaRG cells. Moreover, saturable uptake of reference substrates for the sinusoidal transporters sodium-taurocholate cotransporting polypeptide, OATPs and OCT1 and canalicular secretion of reference substrates for the efflux transporters bile salt export pump and MRP2 were observed. This polarized and functional expression of various sinusoidal and canalicular transporters in HepaRG cells highlights the interest of using these hepatoma cells in xenobiotic transport studies.