Atorvastatin (ATV) is primarily metabolized by CYP3A in the liver to form two active hydroxy metabolites. Therefore, the sequential transport system governed by hepatic uptake and efflux transporters is important for the drug disposition and metabolism. Here, we assessed the interaction of ATV with hepatic uptake transporter organic anion transporting polypeptide (Oatp) and efflux transporter multidrug resistance associated protein 2 (MRP2/Mrp2) in vitro and ex situ using the isolated perfused rat liver (IPRL). Rifampicin (RIF) was chosen as an inhibitor for Oatp in both uptake and IPRL studies. Its inhibitory effects on MRP2 and metabolism were also tested using MRP2-overexpressing cells and rat microsomes, respectively. Our results indicate that RIF effectively inhibits the Oatp-mediated uptake of ATV and its metabolites. Inhibition on MRP2-mediated efflux of ATV was also observed at a high RIF concentration. Compared with ATV alone in the IPRL, the area under the curve(s) (AUC) of ATV was significantly increased by RIF, whereas the AUC of both metabolites were also increased in a concentration-dependent manner. However, the extent of metabolism was significantly reduced, as reflected by the reduced amounts of metabolites detected in RIF-treated livers. In conclusion, inhibition of Oatp-mediated uptake seems to be the major determinant for interaction between ATV and RIF. Metabolites of ATV were subject to Oatp-mediated uptake as well, suggesting that they undergo a similar disposition pathway as the parent drug. These data emphasize the relevance of uptake transporter as being one of the major players in hepatic drug elimination, even for substrates that undergo metabolism.