Rat canalicular membranes contain microdomains enriched in cholesterol and ATP-binding cassette transporters. Cholesterol is known to regulate the activity of transporters. Here, we investigated the effect of membrane cholesterol on the transport kinetics of multidrug resistance-associated protein 2 (MRP2) and of bile salt export pump (BSEP) variants and mutants. MRP2 and BSEP were expressed with baculoviruses in insect cells, followed by vesicle isolation from control and cholesterol-loaded cells (1 mM cholesterol@randomly methylated-β-cyclodextrin) for transport assays. We found that cholesterol stimulates MRP2 transport activity for substrates of different molecular weights: estradiol-17-β-glucuronide (E17βG), prostaglandin E2 (PGE2), cholecystokinin 8 (CCK8), and vasopressin displayed an increase of Vmax and a variable decrease of Km. Kinetics of E17βG showed a sigmoidal shape and a mild cooperativity in Hanes-Woolf plots in control membranes. High cholesterol content shifted E17βG to Michaelis-Menten kinetics. PGE2/glutathione transport followed Michaelis-Menten kinetics irrespective of cholesterol. The MRP2 substrates CCK8 and vasopressin exhibited Michaelis-Menten kinetics independent of membrane cholesterol content. Transport of ochratoxin A was ATP-dependent but was neither mediated by MRP2 nor stimulated by cholesterol. Transport of the two most common BSEP variants p.444V/A showed Michaelis-Menten kinetics irrespective of membrane cholesterol, whereby cholesterol leads to an increased Vmax while Km remains unchanged. The transport activity of the BSEP mutants p.E297G and p.R432T increased at high cholesterol content but did not reach the capacity of normal BSEP. Hence, changing membrane cholesterol content modulates BSEP and MRP2 transport kinetics differently. Cholesterol increases the transport rates of BSEP and MRP2, but with the latter, may also modify the binding site as for E17βG.