Abstract Rhodopsin, a prototypical G protein-coupled receptor, catalyzes the activation of a heterotrimeric G protein, transducin, to initiate a visual signaling cascade in photoreceptor cells. The βγ subunit complex, especially the C-terminal domain of the transducin γ subunit, Gtγ(60–71)farnesyl, plays a pivotal role in allosteric regulation of nucleotide exchange on the transducin α subunit by light-activated rhodopsin. We report that this domain is unstructured in the presence of an inactive receptor but forms an amphipathic helix upon rhodopsin activation. A K65E/E66K charge reversal mutant of the γ subunit has diminished interactions with the receptor and fails to adopt the helical conformation. The identification of this conformational switch provides a mechanism for active GPCR utilization of the βγ complex in signal transfer to G proteins.