The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein a subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single Gα i/o-Loco (GoLoco) motif predicted to represent a second Gα interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with Gαi1, Gαi2, and Gαi3 in their GDP-bound forms. In GTPγS binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by Gαi1. Both regions also stabilize Gαi1 in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF4-. Our results indicate that both RGS12 and RGS14 harbor two distinctly different Gα interaction sites: a previously recognized N-terminal RGS box possessing Gαi/o GAP activity and a C-terminal GoLoco region exhibiting Gαi GDI activity. The presence of two, independent Gα interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple Gα GAP activity.