Membrane transporters of the major facilitator superfamily (MFS) mediate uptake of diverse metabolites, yet the molecular basis of substrate recognition within many bacterial families remains unclear. The anion:cation symporter (ACS) family is conserved from bacteria to humans and includes medically relevant solute carrier transporters, but only few bacterial members have been functionally characterized. Here, we combine genetics, biochemistry, transport assays, and structural biology to define the substrate specificity of five ACS transporters from Escherichia coli . Using systematic growth complementation assays in deletion strains, we assign physiological substrates to each transporter, identifying DgoT, LgoT, and ExuT as specific uptake systems for D-galactonate, L-galactonate, and galacturonate/glucuronate, respectively, while revealing overlapping roles for GarP and GudP in C6 sugar acid uptake. NanoDSF ligand binding assays show highly selective recognition of sugar acids but do not predict transport activity. Proton-coupled uptake was directly demonstrated using reconstituted proteoliposomes, defining strict stereoselectivity of LgoT and DgoT. We determined the 2.2 Å X-ray structure of LgoT in an inward-open conformation, revealing a canonical MFS fold with a conserved but differentially tuned substrate-binding cavity. Comparative structural and sequence analyses across ACS members identify a conserved core for coordination of sugar acid carboxylate and hydroxyl groups, while localized substitutions modulate steric and electrostatic properties to enable discrimination of substrate size and stereochemistry. These results provide a framework for substrate recognition in bacterial ACS transporters and establish LgoT as a structural model for stereo-selective proton-coupled organic anion transport.