The conformational changes at the cytoplasmic ends of transmembrane helices 5 and 6 (TMH5 and TMH6) of thyrotropin-releasing hormone (TRH) receptor type I (TRH-R1) during activation were analyzed by cysteine-scanning mutagenesis followed by disulfide cross-linking and molecular modeling. Sixteen double cysteine mutants were constructed by substitution of one residue at the cytoplasmic end of TMH5 and the other at that of TMH6. The cross-linking experiments indicate that four mutants, Q263C/G212C, Q263C/Y211C, T265C/G212C, and T265C/Y211C, exhibited disulfide bond formation that was sensitive to TRH occupancy. We refined our previous TRH-R1 models by embedding them into a hydrated explicit lipid bilayer. Molecular dynamics simulations of the models, as well as in silico double cysteine models, generated trajectories that were in agreement with experimental results. Our findings suggest that TRH binding induces a separation of the cytoplasmic ends of TMH5 and TMH6 and a rotation of TMH6. These changes likely increase the surface accessible area at the juxtamembrane region of intracellular loop 3 that could promote interactions between G proteins and key residues within the receptor.