SUMMARY Multidrug resistance-associated protein 4 (MRP4) is an ATP-binding cassette (ABC) transporter expressed at multiple tissue barriers where it actively extrudes a wide variety of drug compounds. Overexpression of MRP4 provides resistance to clinically used antineoplastic and antiviral agents, making it a highly attractive therapeutic target for countering multidrug resistance. Here we report the cryo-EM structures of multiple physiologically relevant states of membrane-embedded human MRP4, including complexes between MRP4 and two widely used chemotherapeutic agents and a complex between MRP4 and its native substrate. The structures display clear similarities and distinct differences in the coordination of these chemically diverse substrates and, in combination with functional and mutational analysis, reveal molecular details of the transport mechanism. Our study provides key insights regarding the basis of the unusually broad substrate specificity of MRP4 and constitutes an important contribution towards a general understanding of the versatile ensemble of multidrug transporters.