The hASBT (human apical Na+-dependent bile acid transporter) constitutes a key target of anti-hypercholesterolaemic therapies and pro-drug approaches; physiologically, hASBT actively reclaims bile acids along the terminal ileum via Na+ co-transport. Previously, TM (transmembrane segment) 7 was identified as part of the putative substrate permeation pathway using SCAM (substitute cysteine accessibility mutagenesis). In the present study, SCAM was extended through EL3 (extracellular loop 3; residues Arg254–Val286) that leads into TM7 from the exofacial matrix. Activity of most EL3 mutants was significantly hampered upon cysteine substitution, whereas ten (out of 31) were functionally inactive (<10% activity). Since only E282C lacked plasma membrane expression, EL3 amino acids predominantly fulfill critical functional roles during transport. Oppositely charged membrane-impermeant MTS (methanethiosulfonate) reagents {MTSET [(2-trimethylammonium) ethyl MTS] and MTSES [(2-sulfonatoethyl) MTS]} produced mostly similar inhibition profiles wherein only middle and descending loop segments (residues Thr267–Val286) displayed significant MTS sensitivity. The presence of bile acid substrate significantly reduced the rates of MTS modification for all MTS-sensitive mutants, suggesting a functional association between EL3 residues and bile acids. Activity assessments at equilibrative [Na+] revealed numerous Na+-sensitive residues, possibly performing auxiliary functions during transport such as transduction of protein conformational changes during translocation. Integration of these data suggests ligand interaction points along EL3 via electrostatic interactions with Arg256, Glu261 and probably Glu282 and a potential cation-π interaction with Phe278. We conclude that EL3 amino acids are essential for hASBT activity, probably as primary substrate interaction points using long-range electrostatic attractive forces.