A large number of antimalarial compounds, including the clinical candidate cipargamin, are believed to kill Plasmodium falciparum parasites via inhibition of the parasite protein PfATP4. The characterisation of this transport protein has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Our aim was to optimise a membrane ATPase assay to investigate the function of PfATP4 and its sensitivity to chemical inhibition. We found that cipargamin inhibited the Na+-dependent fraction of ATPase activity measured in P. falciparum membranes from wild-type parasites, with its potency reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the antimalarial compounds in the Medicines for Malaria Venture’s Malaria Box that had previously been shown to disrupt ion regulation in P. falciparum in a cipargamin-like manner; this is consistent with PfATP4 being the direct target of these compounds. Characterisation of the biochemical properties and ion sensitivity of the cipargamin-sensitive membrane ATPase activity yielded data consistent with PfATP4 being a Na+ transporter that is sensitive to physiologically relevant perturbations of pH, but not [K+] or [Ca2+]. With an apparent Km for ATP of 0.2 mM and an apparent Km for Na+ of 16 mM the protein is predicted to operate at less than its half-maximal rate under normal physiological conditions, thus ensuring that it has the capacity to increase its rate in response to an increase in cytosolic [Na+]. In membranes from a cipargamin-resistant PfATP4-mutant line that has an elevated resting cytosolic [Na+], the apparent Km for Na+ is slightly but significantly elevated (i.e. the affinity of the transporter for Na+ is reduced). Our findings shed new light on the biochemical characteristics of an important new drug target and its susceptibility to chemical inhibition.