ABCG2 is a polyspecific membrane transporter of the ATP-binding cassette (ABC) superfamily, which is responsible for the efflux of a wide array of structurally unrelated molecules from mammalian cells. Physiologically, the protein is thought to serve a protective role; ABCG2 is expressed in certain stem cell populations, as well as at “barrier sites” throughout the body. Thus, ABCG2 expression is important from a pharmacokinetic perspective: expression in the gut reduces the bioavailability of orally-administered substrates, whilst its expression at the blood-brain barrier and placenta prevents drug accumulation in the brain and the foetus, respectively. ABCG2 expression can also be problematic in certain cancers, where it causes chemotherapy resistance. Recent structural information about ABCG2 from cryogenic electron microscopy has revealed conformational changes associated with transport. However, there has been little investigation of the driving forces behind the process. ATP hydrolysis alone is traditionally thought to drive ABC transporters, but recent research shows that a transmembrane chemical proton gradient (ΔpH) contributes in certain bacterial ABC multidrug transporters. Therefore, this work aimed to elucidate the effect of ΔpH on ABCG2-mediated transport. First, extensive characterisation of a frequently used fluorescent ABCG2 substrate, Hoechst 33342, revealed that it was unsuitable for this purpose. Despite its efficient transport by ABCG2, the dye’s local pH can drastically affect its fluorescence when associated with lipid and DNA. Therefore, ethidium was favoured as a substrate that does not suffer from these problems. Assays in intact (human) Expi293F cells demonstrated that ABCG2 effluxes ethidium and identified carboxyfluorescein succinimidyl ester (CFSE) as another novel substrate. Experiments with proteoliposomes containing purified inside-out oriented ABCG2 revealed that an interior alkaline ΔpH was necessary and sufficient for ABCG2-mediated ethidium transport. These results are relevant physiologically in the gut where, in acidic regions, enterocytes maintain a substantial ΔpH (interior alkaline) and ABCG2 expression is the highest. The importance of an abnormal ΔpH (interior alkaline) is also well-established in cancer. Furthermore, single-cell expression data suggests that embryonic stem cells might also contain such a proton gradient. Findings from this work are therefore relevant from a drug development perspective, as they point to novel mechanisms by which ABCG2 activity can be modulated in health and disease.