Updated project metadata. Chemotherapy resistance by ABC transporters and secondary transporters is well established. Liver cancer is hard to treat by chemotherapy due to the presence of the multidrug resistance-associated protein 2 (MRP2/ABCC2) in addition to other transporters. The activity of MRP2 can be modulated by phosphorylation of its regulatory domain but also by drug-drug interactions. Here, we have determined the cryo-EM structure of MRP2 from Rattus norvegicus in an autoinhibited state (nucleotide-free) and in the presence of the drug modulator probenecid (uricosuric agent). The autoinhibited conformation is caused by the partially phosphorylated regulatory domain that folds within the transmembrane domain. Combining, in vitro phosphorylation of MRP2, mass spectrometry and kinetics, we show that full phosphorylation of the regulatory domain is required to relieve the autoinhibitory state due to steric clashes between the phosphate groups and the transmembrane domain. The drug bound state, has identified two drug binding sites that has allowed us to propose how probenecid modulates the activity of the transporter in addition to drug-drug interactions. Overall, we have structurally characterised the clinically relevant transporter MRP2, and we provide evidence on its modulation by intracellular kinases but also in the presence of external drugs. Finally, we mapped the mutations that cause Dubin–Johnson syndrome onto the structure and gain an understanding on their contribution in MRP2 dysfunction.