The proton-coupled folate transporter (PCFT, SLC46A1) mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This manuscript addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains (TMDs) with a detailed analysis of the PCFT-E185 residue. D156A, E185A, E232A, R148A, R376A PCFT mutants lost function at pH 5.5 as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for the E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an 8-fold decrease in the [3H]methotrexate (MTX) influx Vmax; the MTX influx Kt was identical to that of wild-type (WT)-PCFT (1.5 µM). Consistent with the intrinsic functionality of E185A-PCFT, [3H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with either nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (~ 38% of WT) for the like-charged E185D at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with the augmentation of a rate-limiting step in the PCFT transport cycle.