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Supplementary data associated with this article can be found in the online version at doi:10.1016/j.csbj.2023.04.025., The Aspergillus niger CexA transporter belongs to the DHA1 (Drug-H+ antiporter) family. CexA homologs are exclusively found in eukaryotic genomes, and CexA is the sole citrate exporter to have been functionally characterized in this family so far. In the present work, we expressed CexA in Saccharomyces cerevisiae, demonstrating its ability to bind isocitric acid, and import citrate at pH 5.5 with low affinity. Citrate uptake was independent of the proton motive force and compatible with a facilitated diffusion mechanism. To unravel the structural features of this transporter, we then targeted 21 CexA residues for site-directed mutagenesis. Residues were identified by a combination of amino acid residue conservation among the DHA1 family, 3D structure prediction, and substrate molecular docking analysis. S. cerevisiae cells expressing this library of CexA mutant alleles were evaluated for their capacity to grow on carboxylic acid-containing media and transport of radiolabeled citrate. We also determined protein subcellular localization by GFP tagging, with seven amino acid substitutions affecting CexA protein expression at the plasma membrane. The substitutions P200A, Y307A, S315A, and R461A displayed loss-of-function phenotypes. The majority of the substitutions affected citrate binding and translocation. The S75 residue had no impact on citrate export but affected its import, as the substitution for alanine increased the affinity of the transporter for citrate. Conversely, expression of CexA mutant alleles in the Yarrowia lipolytica cex1Δ strain revealed the involvement of R192 and Q196 residues in citrate export. Globally, we uncovered a set of relevant amino acid residues involved in CexA expression, export capacity and import affinity., This work was supported by the Strategic Programme UID/BIA/04050/2020 and the project LA/P/0069/2020 granted to the Associate Laboratory ARNET, both funded by Portuguese funds through the FCT-IP. J.A. acknowledges the FCT and the Doctoral Program in Applied and Environmental Microbiology for the PD/BD/150584/2020 PhD grant and a COST Action CA18113 Short-Term Scientific Mission grant (EuroMicropH). M.S.S. acknowledges the Norte2020 for the UMINHO/BD/25/2016 PhD grant with the re ference NORTE-08-5369-FSE-000060. I.S-S. was supported by the program contract FCTUMINHO/Norma transitória from the Legal Regime of Scientific Employment (RJEC).