Transporters responsible for hepatic uptake and biliary clearance (CLBile) of rosuvastatin (RSV) have been well characterized. However, the contribution of basolateral efflux clearance (CLBL) to hepatic and systemic exposure of RSV is unknown. Additionally, the appropriate design of in vitro hepatocyte efflux experiments to estimate CLBile versus CLBL remains to be established. A novel uptake and efflux protocol was developed in sandwich-cultured hepatocytes (SCH) to achieve desired tight junction modulation while maintaining cell viability. Subsequently, studies were conducted to determine the role of CLBL in the hepatic disposition of RSV using SCH from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR(-)) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor elacridar (GF120918). RSV CLBile was nearly ablated by GF120918 in TR(-) SCH, confirming that Mrp2 and Bcrp are responsible for the majority of RSV CLBile. Pharmacokinetic modeling revealed that CLBL and CLBile represent alternative elimination routes with quantitatively similar contributions to the overall hepatocellular excretion of RSV in rat SCH under baseline conditions (WT SCH in the absence of GF120918) and also in human SCH. Membrane vesicle experiments revealed that RSV is a substrate of MRP4 (Km = 21 ± 7 µM, Vmax = 1140 ± 210 pmol/min per milligram of protein). Alterations in MRP4-mediated RSV CLBL due to drug-drug interactions, genetic polymorphisms, or disease states may lead to changes in hepatic and systemic exposure of RSV, with implications for the safety and efficacy of this commonly used medication.