Cryopreserved hepatocytes have been widely employed to characterize drug ADME features in pharmaceutical industry. The aim of this study is to characterize the uptake and efflux transporters in human cryopreserved hepatocytes. More than 30 lots of human cryopreserved hepatocytes were tested for the ability to form bile canaliculi network upon culturing in the sandwich configuration. Briefly, cryopreserved hepatocytes were thawed and seeded on a 24-well BD BioCoatTM Collagen I plate at density of 400,000 cells/well. After overnight incubation, the cells were overlaid with 0.5mL of ice cold 0.25mg/mL BD MatrigelTM prepared in Williams’ E media. The culture media was replaced every 24 hrs. On day 5 post culture, cells were rinsed with regular HBSS, and for efflux assays, 5 μM CDFDA or 5 μM CLF in regular HBSS with or without specific transporter inhibitors was added into cell culture. Bile canaliculi accumulation of fluorescence compounds was assessed by fluorescence microscope. For uptake assay, 1 μM of taurocholic acid, 2 μM rosuvastatin prepared in regular HBSS buffer was added into cell culture. The accumulation of these probe substrates inside of hepatocytes were evaluated at 2min, 10min and 15min post treatment. In sandwich cultured configuration, platable cryopreserved hepatocytes can form bile canaliculi network which was evidenced by bile accumulation of two fluorescence substrates, CDF and CLF, which was sufficiently inhibited by MRP2 and BSEP inhibitor respectively. The results demonstrate that plated cryopreserved hepatocytes maintain the functionality of major ABC transporters in sandwich culture. In addition, the dramatic uptake activity of selected substrates demonstrates the functionality of major hepatic uptake transporters, e.g. NTCP and OATP. Functional hepatic uptake and efflux transporters are detected in cryopreserved human hepatocytes in sandwich culture, which provide a useful tool to characterize the drug-drug interaction or drug induced hepatic toxicity during drug discovery.