Organic anion transporting polypeptide 1c1 (Oatp1c1) is a high-affinity T4 transporter expressed in brain barrier cells. To identify Oatp1c1 amino acid residues critical for T4 transport, consensus membrane topology was predicted and a three-dimensional Oatp1c1 structure was generated using the known structures of major facilitator superfamily (MFS) transporters, glycerol 3-phosphate transporter, lactose permease, and the multidrug transporter Escherichia coli multidrug resistance protein D as templates. A total of nine amino acid mutations were generated based on amino acid conservation, localization to putative transmembrane domains, and side chain functionality. Mutant constructs were transiently transfected into human embryonic kidney 293 cells and assessed for plasma membrane localization and the capacity to transport substrate 125I-T4. Wild-type Oatp1c1, R601S, P609A, W277A/W278A, W277F/W278F, G399A/G409A, and G399L/G409L were all expressed at the plasma membrane. Wild-type Oatp1c1 and W277F/W278F displayed biphasic T4 transport kinetics, albeit the mutant did so with an approximately 10-fold increase in high-affinity Michaelis constant. The W277A/W278A mutation abolished Oatp1c1 T4 transport. G399A/G409A and G399V/G409V mutants displayed near wild-type activity in an uptake screen but exhibited diminished T4 transport activity at high-substrate concentrations, suggesting a substrate binding site collapse or inability to convert between input and output states. Finally, transmembrane domain 11 mutants R601S and P609A displayed partial T4 transport activity with significantly reduced maximum velocities and higher Michaelis constant. Arg601 is functionally strongly conserved with members of the MFS whose structures and function have been extensively studied. These data provide the experimental foundation for mapping Oatp1c1 substrate binding sites and reveal evolutionary conservation with bacterial MFS transporter members.