The human ABC transporter ABCC3 (also known as MRP3) transports a wide spectrum of substrates, including endogenous metabolites and exogenous drugs. Accordingly, it participates in multiple physiological processes and is involved in diverse human diseases such as intrahepatic cholestasis of pregnancy, which is caused by the intracellular accumulation of bile acids and estrogens. Here, we report three cryogenic electron microscopy structures of ABCC3: in the apo‐form and in complexed forms bound to either the conjugated sex hormones β‐estradiol 17‐(β‐D‐glucuronide) and dehydroepiandrosterone sulfate. For both hormones, the steroid nuclei that superimpose against each other occupy the hydrophobic center of the transport cavity, whereas the two conjugation groups are separated and fixed by the hydrophilic patches in two transmembrane domains. Structural analysis combined with site‐directed mutagenesis and ATPase activity assays revealed that ABCC3 possesses an amphiphilic substrate‐binding pocket able to hold either conjugated hormone in an asymmetric pattern. These data build on consensus features of the substrate‐binding pocket of MRPs and provide a structural platform for the rational design of inhibitors. Synopsis The human ABC transporter ABCC3/MRP3 transports diverse metabolites and drugs in multiple physiological processes. Here, both structural analysis and biochemical assays of ABCC3 are used to refine the common features of substrate‐binding pockets in multidrug resistance proteins. Three cryo‐EM structures of human ABCC3 are reported: the apo‐, E217βG‐ and DHEAS‐bound forms. The two substrate‐bound ABCC3 complexes display a similar V‐shaped binding pocket, the hydrophobic center of which accommodates the steroid nuclei, whereas the hydrophilic patches stabilize the two conjugation groups of the substrate. The conjugation groups confer substrate specificity, and are coordinated by the conserved positively charged residues. A common substrate‐binding feature is proposed for all MRPs. This cryo‐EM study uses the location of two substrates to reveal a common steroid‐binding pocket within the human ABCC3 protein.